mycobacterium tuberculosis - host-cell interactions in the

Research Center Borstel

Leibniz-Center for Medicine and Biosciences

Priority Area Infections

Program Director: Prof. Dr. Ulrich Schaible

Cellular Microbiology Group

Mycobacterium tuberculosis -

Host-Cell Interactions in the Phagosome

Dissertation

for Fulfillment of Requirements

for the Doctoral Degree

of the University of Lübeck

from the Department of Natural Sciences

Submitted by

Anna Christina Geffken

from Büschelskamp/Scheeßel

________________________________________________________________

First referee: Prof. Dr. Ulrich E. Schaible

Second referee: Prof. Dr. Jan Rupp

Date of oral examination: 20.01.2016

III

Content

ABSTRACT ............................................................................................................... 8

1 INTRODUCTION .............................................................................................. 10

1.1 Tuberculosis ................................................................................................ 10

1.1.1 History of Tuberculosis ........................................................................... 10

1.1.2 Epidemiology of Tuberculosis ................................................................. 11

1.1.3 The disease generalities......................................................................... 12

1.1.3.1 Stages of TB ................................................................................... 12

1.1.3.2 Therapy ........................................................................................... 13

1.1.3.3 Vaccines ......................................................................................... 13

1.2 Mycobacterium tuberculosis ...................................................................... 13

1.2.1 Microbiology of the Mycobacterium tuberculosis complex ...................... 13

1.2.2 The process of M. tuberculosis infection ................................................ 14

1.3 The role of innate immunity in Tuberculosis disease............................... 15

1.3.1 Cells of the innate immune system ......................................................... 15

1.3.2 The role of phagocytes in innate immunity ............................................. 16

1.3.3 Pattern recognition receptors of mycobacterial antigens ........................ 16

1.3.4 The cell biology of phagocytosis in professional phagocytes .................. 18

1.4 Host-pathogen interactions in the phagosome ......................................... 24

1.4.1 The role of cell envelope and secreted proteins in M. tuberculosis

virulence ............................................................................................................... 24

1.4.2 The role of cell envelope lipids in M. tuberculosis virulence ................... 26

1.5 Objectives .................................................................................................... 30

2 MATERIAL AND METHODS ............................................................................ 31

2.1 Material ......................................................................................................... 31

2.1.1 Consumables ......................................................................................... 31

2.1.2 Chemicals .............................................................................................. 32

2.1.3 SiRNA .................................................................................................... 34

2.1.4 Lipids ...................................................................................................... 34

IV

2.1.5 Antibodies and dyes ............................................................................... 35

2.1.6 Cell-lines ................................................................................................ 35

2.1.7 Bacteria.................................................................................................. 35

2.1.8 Hardware ............................................................................................... 35

2.1.9 Software ................................................................................................. 37

2.2 Methods ....................................................................................................... 38

2.2.1 Isolation and purification of lipid coated bead phagosomes from

macrophages for mass spectrometry analysis ..................................................... 38

2.2.1.1 Cell strains and culture conditions .................................................. 38

2.2.1.2 Cell counting ................................................................................... 38

2.2.1.3 Coating of beads with M. bovis BCG cell-wall lipid Trehalose-6,6’-

dimycolate ....................................................................................................... 38

2.2.1.4 “Infection” of macrophages with lipid coated beads ........................ 39

2.2.1.5 Isolation and purification of lipid coated bead phagosomes from

macrophages .................................................................................................... 39

2.2.1.6 Purification of lipid coated bead phagosomes via sorting by FACS 40

2.2.1.7 Quality control and quantification of lipid coated bead phagosomes 41

2.2.2 Methods for proteomic analysis ............................................................. 42

2.2.2.1 Modifications to chapter 2.2.1 for proteomic analysis ...................... 42

2.2.2.2 1D-PAGE ........................................................................................ 42

2.2.2.3 Gel-based liquid chromatography coupled to mass spectrometry

(GeLC-MS) ....................................................................................................... 42

2.2.2.4 Data Analysis .................................................................................. 44

2.2.3 Methods for evaluation of TDM interaction partners ............................... 44

2.2.3.1 Cell culture ...................................................................................... 44

2.2.3.2 “Infection” of macrophages with lipid coated beads ........................ 44

2.2.3.3 Infection of macrophages with M. tuberculosis ............................... 44

2.2.3.4 Lysosomal labeling ......................................................................... 45

2.2.3.5 Immunofluorescence staining ......................................................... 45

2.2.3.6 Gene knock down by small interference RNA ................................. 46

2.2.3.7 Western blot.................................................................................... 46

2.2.3.8 Colony forming unit assay ............................................................... 48

2.2.4 Methods for lipidomic analysis ............................................................... 48

2.2.4.1 Modifications to chapter 2.2.1 for lipidomic analysis ....................... 48

V

2.2.4.2 Lipid extraction from isolated and purified lipid coated bead

phagosomes ..................................................................................................... 49

2.2.4.3 Derivatization of cholesterol ............................................................ 50

2.2.4.4 µLC-FT-ICR-MS .............................................................................. 50

2.2.4.5 ESI Qq-TOF-MS .............................................................................. 51

2.2.4.6 Data processing and analysis.......................................................... 52

2.2.4.7 Statistical analysis and graphic representation ................................ 53

3 RESULTS ......................................................................................................... 54

3.1 Method development: Isolation and purification of bead phagosomes for

mass spectrometry analysis ................................................................................. 54

3.1.1 Quality control of purified bead phagosomes.......................................... 55

3.1.2 Purification of bead phagosomes via sorting by FACS ........................... 56

3.2 The TDM bead phagosome proteome ........................................................ 58

3.2.1 Introduction of selected proteins and STRING analysis .......................... 67

3.3 Evaluation of TDM interaction partners ..................................................... 69

3.3.1 Evaluation of the presence of candidate proteins on control bead, TDM

bead and M. tuberculosis phagosomes by immunofluorescence staining............. 69

3.3.2 Evaluation of the presence of candidate proteins on isolated and purified

control and TDM bead phagosomes by Western blot ........................................... 81

3.3.3 Evaluation of the role of candidate proteins for intracellular survival of M.

tuberculosis by small interfering RNA/CFUs analysis ........................................... 82

3.4 A role for the β-actin cytoskeleton in TDM-mediated inhibition of

phagosome maturation ......................................................................................... 84

3.5 The lipid coated bead phagosome lipidome ............................................. 89

4 DISCUSSION .................................................................................................... 95

4.1 Resume ........................................................................................................ 95

4.2 Method development: Isolation and purification of bead phagosomes

from macrophages for mass spectrometry analysis........................................... 97

VI

4.2.1 Quantification of phagosomal proteins by spectral counting is a label-free

and semi-quantitative approach ......................................................................... 100

4.2.2 Evaluation of proteomics data by immunofluorescence staining and

Western blot revealed contradicting results ........................................................ 100

4.3 TDM induced changes on the bead phagosome proteome ................... 102

4.3.1 TDM bead phagosomes accumulate proteins of the annexin superfamily .

............................................................................................................. 107

4.3.2 TDM bead phagosomes accumulate proteins involved in vesicle fusion ....

............................................................................................................. 109

4.4 The actin cytoskeleton is involved in M. tuberculosis-mediated inhibition

of phagosome maturation ................................................................................... 111

4.4.1 β-actin accumulates on TDM bead and M. tuberculosis phagosomes . 112

4.4.2 Removal of β-actin drives M. tuberculosis into a functional phago-

lysosome ............................................................................................................ 113

4.4.3 Proteins regulating actin treadmilling are important for survival of M.

tuberculosis in macrophages but not the WASH-complex .................................. 113

4.5 TDM-mediated inhibition of phagosome maturation changes the lipid

composition of bead phagosomes ..................................................................... 116

4.6 Conclusion: TDM-mediated changes of the phagosomal proteome and

lipidome – potential interaction partners or innocent bystanders? ................ 120

4.7 Perspectives .............................................................................................. 122

LITERATURE ........................................................................................................ 124

SUPPLEMENTARY MATERIAL ........................................................................... 141

LIST OF FIGURES ................................................................................................ 180

LIST OF TABLES.................................................................................................. 182

LIST OF ABBREVIATIONS AND SYMBOLS ....................................................... 183

PUBLICATIONS AND CONFERENCES ............................................................... 187

VII

ACKNOWLEDGEMENTS .................................................................................... 1888

ERKLÄRUNG .................................................................................................... 18989

Abstract

8

Abstract

Worldwide, Tuberculosis is the prime human “killer” caused by a single bacterial

pathogen species, Mycobacterium tuberculosis. Still, the interactions between M.

tuberculosis and its predominant host-cell, the macrophage, are far from being fully

understood at the molecular level. Phagocytosed particles become enclosed inside

phagosomes which, under normal conditions, subsequently mature to phago-

lysosomes by successive fusion and fission events upon interaction with endo- and

lysosomes. M. tuberculosis however, stalls phagosome maturation at an early

phagosomal stage to create a niche for its survival and proliferation.

For that purpose, M. tuberculosis requires virulence factors impeding phagosomal

maturation. Positioned at the surface of mycobacteria, cell-wall glycolipids represent

the molecular forefront of macrophage-M. tuberculosis interactions. Lipidome-based

studies revealed glycolipid, trehalose-6,6’-dimycolate (TDM) as important contributor

to inhibition of phagosome maturation, but the actual virulence function of TDM and its

interaction partners remain elusive. To study the function of single M. tuberculosis lipid

species in phagosome biogenesis, we established a “lipid coated bead model”. Upon

phagocytosis, lipid coated bead phagosomes were purified and analyzed by mass

spectrometry for their protein and lipid compositions.

By a systems-biology approach combining lipidomics, proteomics and RNAi studies,

we revealed that (i) the TDM bead phagosome proteome is different compared to

control ones, (i) the actin cytoskeleton represents an indirect TDM target for inhibition

of phagosome maturation and (iii) differential phagosomal membrane lipid

compositions by TDM interaction may promote association of actin cytoskeleton

nucleation promoting factors. To conclude, we identified putative target structures to

interfere with TDM virulence function and intracellular survival of M. tuberculosis.

Zusammenfassung

Tuberkulose ist, nach HIV/AIDS, die zweithäufigste Infektionskrankheit des

Menschen. Die WHO schätzt, dass circa ein Drittel der Weltbevölkerung latent mit TB

infiziert ist und jedes Jahr etwa 9 Millionen Menschen an TB erkranken und 1.5

Millionen daran sterben. Trotzdem ist die Interaktion zwischen Mycobacterium

tuberculosis und der favorisierten Wirtszelle, den Makrophagen, auf molekularer

Ebene nicht genau verstanden. Wenn Makrophagen nicht intrazellulär lebende

Bakterien phagozytieren, werden diese durch die Reifung des so entstandenen

Phagosomes und der Fusion mit Lysosomen schrittweise verdaut und auf diese Weise

9

unschädlich gemacht. Als intrazelluläres Bakterium jedoch, hat sich M. tuberculosis

auf das Überleben in Phagozyten spezialisiert und wird von den Zellen des

angeborenen Immunsystems nicht zerstört.

Um dies zu erreichen, besitzt M. tuberculosis ein breites Spektrum an Virulenz-

faktoren. Zu diesen zählen, unter anderem, die einzigartigen Lipide der mykobak-

teriellen Zellwand. Glycolipide wie Trehalose-6,6´-dimykolat (TDM) befinden sich an

der vorderster Front, wenn Mycobakterien auf ihre Wirtszelle treffen. Vorherige

Studien konnten zeigen, dass TDM für die Inhibition der Phagosomen Reifung eine

Schlüsselrolle spielt. Trotzdem ist die molekulare Virulenzfunktion von TDM nicht

geklärt.

Um die Funktion einzelner Lipidspezies von M. tuberculosis zu untersuchen, haben

wir ein „lipid coated bead model“ etabliert, welches die Infektion von Wirtszellen durch

Mykobakterien nachahmen soll. In diesem Modell werden TDM ummantelte Beads

von Makrophagen phagozytiert und die so entstandenen Bead Phagosomen isoliert,

aufgereinigt und mit Hilfe massenspektrometrischer Analysen auf ihre Protein- und

Lipid-Zusammensetzung untersucht.

Unsere Untersuchungen ergaben, (i) dass sich das Proteom von TDM-Bead-

Phagosomen von Kontroll-Bead-Phagosomen unterscheidet, (ii) dass das Aktin

Zytoskelett eine Zielstruktur darstellt, die durch die Interaktion der Zelle mit TDM

beeinflusst wird, um die Phagosomen Reifung zu hemmen und (iii) dass Lipide in der

Phagosomen-Membran direkte TDM-Interaktionspartner darstellen, welche TDM

manipuliert um Nukleations-Promotionsfaktoren für die Bildung des Aktin Zytoskeletts

am Phagosom zu rekrutieren.

Introduction

10

1 Introduction

1.1 Tuberculosis

1.1.1 History of Tuberculosis

“Es geht so nicht weiter. Ihre Frau Tochter, wenn nicht etwas geschieht, das sie der

Einsamkeit und dem Schmerzlichen ihres nun seit Jahren geführten Lebens entreißt,

wird schnell hinsiechen. Eine Disposition für Phthisis war immer da…[…].“

Effi Briest, Theodor Fontane

Phthisis, Schwindsucht, consumption, scrofula, Pott's disease or white plaque, are

several descriptive names for one single infectious disease, nowadays termed

Tuberculosis (TB). TB is an aerosol-transmitted infectious disease of the lung caused

by bacterial pathogens of the Mycobacterium tuberculosis complex (MTBC).

In 1984, John Hayman hypothesized that mankind has co-evolved together with

mycobacterial pathogens since 150 million years [1]. However, only with increasing

population size based on the industrial revolution, during the 19th and early 20th

century, TB became pandemic. In the late 19th century, 70 % to 90 % of the urban

population of Europe and North America were infected and about 80 % of those

individuals with active TB died [2]. In 1882, the knowledge about TB changed

dramatically when Robert Koch held a presentation entitled “Die Aetiologie der

Tuberkulose” [3]. Robert Koch identified the tubercle bacillus as etiological agent of

TB. Based on these findings, in 1921 Albert Calmette and his associate Camille Guérin

developed the first vaccine against TB by attenuating Mycobacterium bovis.

Consequently, in the next decades, the BCG (Bacille Calmette-Guérin) vaccine was

used to vaccinate millions of infants worldwide. Despite the knowledge of the

etiological agent of TB, treatment was limited to inpatient sanatoria where patients

were treated by rest, fresh air, sunlight and a rich diet [4]. Only in 1944 and 1946, TB

treatment became achievable when the first antibiotics effective against M.

tuberculosis, streptomycin and PAS, were discovered [5]. Consequently, the number

of TB cases declined in Europe due to improved socioeconomic conditions, better

nutrition, hygiene and surveillance sequestration of TB patients but also as a results

of successful antibiotic treatment [6].

However, from 1985 on, TB experienced a renaissance primarily in developing, but

also to some extent in industrialized countries. This was largely due to the HIV/AIDS

pandemic [7]. For the period 1990-1999, the World Health Organization (WHO)

Introduction

11

predicted an increase in annual global incidence from 7.5 million to 10.2 million new

cases [8]. Consequently, the WHO established a global framework for TB control

termed DOTS (directly observed therapy, short course) to improve case detection,

monitoring and supervision of drug intake [9]. Additionally, in 1998, the Stop TB

initiative was established as a global plan for TB control. In 2006 this was further

extended by establishing the Stop TB partnership and to the Global Plan to Stop TB

2006-2015. This plan aimed to halve TB prevalence and mortality compared with 1990

levels by 2015 and to achieve TB elimination by 2050. It can be quoted as success

that between 1990-2010 global TB mortality rates fell by around 40 % [10].

1.1.2 Epidemiology of Tuberculosis

Still, TB remains the second leading cause of death from a single infectious agent

worldwide after HIV [11]. The WHO estimates that one third of the human population

is latently infected. In the year 2013, globally there were an estimated 9 million cases

and 1.5 million deaths [11]. However, the current situation is torn: the WHO classifies

22 high burden countries that account for over 81 % of the world’s TB cases [10].

Among those are the South East Asian region, the African Region and the Western

Pacific Region. In stark contrary are the developed countries of the Americas and the

European Region with only 3 % and 4 % of incident cases in 2013, respectively (Figure

1.1) [11].

Introduction

12

Figure 1.1: Global trends in estimated rates of TB incidence in 2013. Modified from [11]. The

WHO classifies 22 high burden countries accounting for 81 % of the worlds TB cases. These in-

clude the South East Asian Region, the African Region and the Western Pacific Region. To the

contrary, the Americas and the European Region harbour 3 % and 4 % of all cases, respectively.

HIV remains a very important risk factor to develop acute TB. As within the 9 million

incident cases in 2013, 13 % were HIV co-infected. Another alarming scenario is the

increase in multi- and extensively-drug-resistant (MDR/XDR)-TB, primarily in countries

with functional but substandard heath care systems. Here repeated inadequate or

incomplete drug therapies are likely to be the cause of the emergence of drug re-

sistance in M. tuberculosis. Most of these cases occur in Russia, China and India [10].

1.1.3 The disease generalities

1.1.3.1 Stages of TB

Infection with TB primarily occurs by inhalation of pathogen containing aerosols (Ø

<5 µm) originating from humans with active pulmonary TB expelled by coughing or

sneezing. As few as 1-3 bacteria are sufficient to cause infection [11]. In 70 % of cases,

TB manifest as pulmonary disease [12]. However, extra-pulmonary TB occurs

frequently and aside from the lung, practically every part of the human body can be

infected.

Figure 1.2: Stages of M. tuberculosis infection. Modified from [13]. After TB infection, 95 % of

cases develop a latent infection. Only 5 % progress to active infection directly. Co-infection with

HIV is the most common risk factor to drive latent to active TB infection.

After pathogens have entered the lung, TB pathogenesis can progress in two different

ways distinguished by the elapsed time between infection and disease onset: primary

or post-primary TB. 5 % of cases develop primary active disease, while 95 % do not

proceed to active infection but become infected to remain asymptomatic, termed latent

TB (Figure 1.2) [13]. Even decades after infection and containment of the disease, in

Introduction

13

5-10 % of cases, latent TB can be reactivated termed post-primary TB [14]. The most

prominent risk factor to progress from latent to active TB today is still HIV co-infection.

However diabetes, tumor necrosis factor α (TNF-α) antagonist therapies of

autoimmune disease patients and smoking are other important risk factors [15]–[17].

1.1.3.2 Therapy

TB can be treated with antibiotics. However, single drug therapy is not recommended

since this treatment will inevitably promote drug resistance [18]. Thus multi-drug

therapy is standard and to date there are 28 different approved antibiotic species

classified as first-line and second-line drugs. First-line drugs are the core TB treatment

and have bactericidal activity while second-line drugs are rather bacteriostatic [18].

The WHO recommended regimen for treatment of active drug-susceptible TB is

divided to two phases: an initial phase (intensive phase) of two months comprising four

first-line drugs followed by a four months treatment (stabilisation phase) with

combination of two first-line drugs [19],[23]. With this, cure rates of more than 90 %

are achieved [21]. An emerging problem is the development of MDR- and XDR-TB

because the drugs applied today are in use since 1960 and have not been properly

used in many settings and regions. Evidently, new TB drugs are urgently needed. In

addition, new attempts should be made to treat TB that must include better

understanding of the host-pathogen interaction early in infection. That way, host-

directed therapies can be developed.

1.1.3.3 Vaccines

Up to date, there is no vaccine that reliably prevents TB. Developed in 1921, the BCG

vaccine is effective only in the prevention of severe disease as meningitis and military

TB in children under 5 years [18]. While in low-risk transmission countries such as

Germany BCG vaccination is no longer advised, in high burden countries the WHO

still recommends BCG vaccination for neonates [11],[19]. Regardless, overall the

vaccine has an estimated efficacy of approximately 50 % for the prevention of TB and

is thus not expected to have any significant impact in reducing global TB incidence

[18],[22]. Thus new and more efficient TB vaccines must be developed.

1.2 Mycobacterium tuberculosis

1.2.1 Microbiology of the Mycobacterium tuberculosis complex

Within the kingdom of bacteria, M. tuberculosis belongs to the phylum of

Actinobacteria and is further classified in the family Mycobacteriaceae and the genus

Introduction

14

Mycobacterium [23]. Due to the unique mycobacterial cell-wall they cannot truly be

classified as Gram-positive. Mycobacteria are rod-shaped and non-motile with a size

of 0.2 to 0.6 µm by 1 to 10 µm. In liquid culture their growth is pleomorphic as they can

occur as single rods or as multicellular and branched filaments.

Human and animal pathogens of the Mycobacterium tuberculosis complex are

classified as slow-growers (generation time >24 h) while avirulent species such as e.g.

Mycobacterium smegmatis are fast-growers (generation time >3-4 h) and are mostly

found in environmental habitats such as water or soil. MTBC bacteria are obligate

pathogens of mammals and comprises seven different species: M. tuberculosis, M.

bovis (ssp. bovis and caprae), M. africanum, M. microti, M. canetti und M. pinnipedii

[24]. M. tuberculosis is the most notorious pathogen in humans [10]. M. canetii and M.

africanum (clade I and II) are also human pathogens but are primarily isolated from

African patients. M. bovis is known as the causative agent of TB in cattle but has a

wide host spectrum including humans, domestic or wild bovines. M. caprae, M.

pinipedii are able to infect goats, deer and seals, respectively [25],[26].

1.2.2 The process of M. tuberculosis infection

The succession of events following entry of M. tuberculosis to the lung has been

extensively studied and therefore is well established. After inhalation into the airways

and attachment to lung airway epithelia and surfactant, patrolling alveolar

macrophages or dendritic cells (DCs) of the innate immune system are the first cells

to encounter and phagocytose the pathogen [27].

Figure 1.3: The immunological process following M. tuberculosis infection in the lung.

Modified from [28]. See text for details.

This induces a localized pro-inflammatory response that leads to the recruitment of

mononuclear cells as DCs, neutrophils and monocytes from neighbouring blood

vessels. Onwards, DCs engulf bacteria or bacterial components, circulate to the

Introduction

15

draining lymph nodes and present mycobacterial antigens via the major

histocompatibility complex (MHC) class I or class II or CD1 molecules to naive T-cells

[29]. T-cells then migrate to the lungs to orchestrate anti-infection immunity, thereby

enhancing the antibacterial activity of macrophages by releasing activating cytokines,

such as interferon-γ (IFN-γ) and TNF-α. This T-helper 1 response is considered to

result in arrest of the infection in granuloma or complete clearance [30]. The bacteria

can persist within granulomas for years to decades in a dormant stage leading to latent

TB. Granulomas represent an organized structure composed of macrophages, giant

foamy cells (derived from macrophages), T- and B-cells as well as blood vessels.

Furthermore granulomas become encapsulated by a fibrotic and often calcified matrix.

Latent TB is therefore described as a state of equilibrium between bacterial growth

and immune control, in which maintained immune responses sequester the infection

locally, thus permitting host-controlled persistence [31]. Exacerbating lung pathology

is limited via regulatory T-cells and probably myeloid suppressor cells by the release

of anti-inflammatory cytokines as interleukin-10 and transforming growth factor-β.

The latent TB stage is asymptomatic. In contrast, active primary and post-primary TB

encompasses a heterogeneous range of symptoms depending on various factors as

the site of infection, co-morbidity or age [32]. Independent from the site of infection,

the clinical presentation of active TB shares systematic symptoms as fever, malaise,

weakness and weight loss [18]. Symptoms of pulmonary TB are cough, hemoptysis

and radiological abnormalities such as lung cavities or densities [33].

1.3 The role of innate immunity in Tuberculosis disease

1.3.1 Cells of the innate immune system

In humans, as all mammals, immunity to pathogens is mediated by the innate and

adaptive immune system. Innate immunity refers to intrinsic and rather nonspecific

defence mechanisms while acquired immunity matures over time to become highly

antigen specific. However, both types of immune responses are the result of cells

circulating the blood and lymphatic fluid [23]. All cells comprising the immune defense

derive from a common progenitor termed (hematopoietic) stem cell. Stem cells locate

in the bone marrow. Through the influence of distinct cytokines they differentiate to

myeloid and lymphoid progenitor cells and erythroid megakaryocytes [34]. Myeloid

progenitors give rise to innate immune cells such as myeloid DCs, macrophages,

neutrophils, eosinophils, basophils and mast-cells. Lymphoid progenitors mature to T-

Introduction

16

and B-lymphocytes, NK-cells (natural killer cells) as well plasmacytoid DCs yielding

the acquired immune system.

1.3.2 The role of phagocytes in innate immunity

When a pathogen invades a host, specialized white blood cells called phagocytes of

the innate immune system are the first cells to deal with the invader. Phagocytes are

at the starting point of the innate and adaptive immune response because they are

able to (i) recognize, engulf and destroy microbes, (ii) release pro-inflammatory

cytokines and chemokines to attract inflammatory innate cells but also those of the

adaptive immune system and (iii) to process and present foreign antigens for

presentation to T-cells. The term phagocyte has its origin in the ancient Greek terms

“phagein; eating” and “cytos; cell” [23]. The process of phagocytosis is defined as the

ingestion of particles larger than 0.5 µm and was discovered more than 100 years ago

by Ilja Mechnikov [35]. In mammals, three different professional phagocytes can be

distinguished: macrophages, neutrophils and DCs. The macrophage is the central cell

in innate immunity against M. tuberculosis.

Phagocytosis is a relatively specific process because it involves recognition and

binding of “prey” by receptors on the phagocyte surface [36]. Recognition of foreign

particles by professional phagocytes is facilitated by germline encoded pattern

recognition receptors (PRRs) specific for pathogen associated molecular pattern

(PAMP) [37]. PAMPs are molecules absent from higher organisms and therefore serve

as identifiers of pathogens for the innate immune system. PRRs that sense PAMPs of

Mycobacteria are of various families and include the Toll-like receptors (TLRs), the

nucleotide oligomerization domain (NOD)-like receptors (NLRs), scavenger receptors

(SR) and C-type lectin receptors (CLRs). Phagocytes also express opsonic receptors

that bind pathogens decorated by opsonins as immunoglobulins or components of the

complement cascade. These comprise the Fcγ (FcγR) and complement receptors

(CRs). Many of these interactions mediate either pro- or anti-inflammatory signaling

events or phagocytosis, or both.

1.3.3 Pattern recognition receptors of mycobacterial antigens

TLRs are archetypal PRRs located at plasma or endosomal membranes of innate as

well as adaptive immune cells. Upon ligand binding and receptor dimerization, several

adaptor proteins transport the signal into the nucleus. Subsequently, the NF-ĸB

transcriptional regulator induces the production of pro-inflammatory cytokines and

chemokines. Several TLRs were described to sense mycobacterial PAMPs:

Introduction

17

Heterodimers of TLR2/TLR6 and TLR2/TLR1 have reported to sense triacylated and

diacylated lipoproteins, respectively [41],[42]. Other reported ligands for TLR2 are

mycobacterial cell-wall lipids as trehalose-6,6-dimycolate (TDM), lipomannan (LM)

and phosphatidylinositol mannosides (PIMs) [43],[44]. Furthermore, unmethylated,

mycobacterial DNA with CpG motifs was shown to be recognized by phagosomal

TLR9 [41].

M. tuberculosis can also interact with receptors of the NLR family [42]. NOD-like

receptors are intracellular sensors of PAMPs since they are primarily localized in the

cytoplasm of monocytes and macrophages. NOD2 senses muramyl dipeptide (MDP)

and synergizes with TLR2 to induce an inflammatory response to M. tuberculosis.

Additionally the NLRP3 recognizes M. tuberculosis secreted ESAT6 to promote

inflammasome activation [43].

Lung monocytes/macrophages and DCs express SRs on their plasma membrane. SR

sub-group A consists of SR-AI, SR-AII and MARCO, while SR sub-group B consists

of SR-B1 and CD36 [44]. Of these, the macrophage receptor with collagenous

structure (MARCO) and class A SR types I and II (SR-A) have been shown to bind

acetylated low-density lipoprotein. MARCO was recently discovered to bind TDM and

thereby to tether M. tuberculosis to macrophages, activate the TLR4/CD14 signalling

pathways and initiate phagocytosis [39]. SR-A also senses TDM but rather induces

anti-inflammatory actions. CD36 was shown to be involved in the uptake of

Mycobacteria by class B Drosophila macrophage-like cells in crosstalk with TLR2 [45].

CTLs represent another family of PRRs sensing mycobacterial PAMPS, comprising

the mannose receptor (MR), Dectin-1/2, the macrophage inducible Ca2+-dependent

(C-type) lectin (Mincle), MCL and the dendritic cell-specific intercellular adhesion

molecule-3-grabbing non-integrin (DC-SIGN). The MR, a transmembrane protein

expressed on tissue and alveolar macrophages as well as DCs, binds various

mycobacterial antigens such as lipoarabinomannan (LAM), PIM, arabinomannans,

mannans and mannose-containing proteins and induces phagocytosis [52],[53].

Dectin-1 is expressed in macrophages, DCs and neutrophils and initiates phagocytosis

by sensing mycobacterial α-glucans [48]. Mincle was recently discovered to be a key

CTL in recognition of TDM [49]. Moreover, also MCL, an FcRγ-coupled activating

receptor, was shown to respond to TDM [50]. DC-SIGN is present on the membrane

of DCs, tissue and alveolar macrophages. As key receptor for M. tuberculosis, it

recognizes antigens as LAM, 19 kDa antigen molecules, ManLAM, LM,

Introduction

18

arabinomannan, mannosylated glycoproteins, PIMs and α-glucans [51]–[54]. Of the

soluble collectins from the CLRs family, the soluble mannose binding lectin (MBL) and

surfactant proteins A and D (SP-A/D) are able to sense molecules of M. tuberculosis.

SP-A and SP-D, produced by the respiratory epithelium, bind lipoarabinomannans

(LAM) and glycoproteins of the mycobacterial cell-wall thereby promoting

phagocytosis and enhanced phago-lysosomal fusion [55]. Synthesized in the liver and

circulating the extracellular space, MBL bind to glycans such as D-mannose or L-

fucose [56]. Thereby complement is directly activated and linked to the MBL coat via

the classical lectin pathway to facilitate CR binding and phagocytosis.

Of the opsonic receptors, the complement receptor 3 (CR3), expressed in neutrophils,

monocytes, NK-cells and macrophages, is involved in the complement pathway for the

binding of opsonized Mycobacteria [57]. Complement opsonins such as C3b and C3b

bind the antigen 85C (AG85C) of M. tuberculosis, tether the bacilli to the membrane

of phagocytes and activate both classical and/or alternative complement pathways.

CR3 also directly interacts with the bacilli since it is able to bind to LAM, β-β-glucans,

mannose and PIMs [58]–[60]. Further, the family of Fc-receptors bind antibodies which

complex M. tuberculosis and antigens thereof. These receptors bind the Fc-part of

immunoglobulin antibodies. In the context of M. tuberculosis infection, Fc-receptors of

the γ-type have the most important role since, in response to mycobacterial infection,

they mediate the rapid ingestion of IgG opsonized particles. FcγR are found on the

surface of macrophages, DCs, neutrophils, B- and NK-cells. Antigen-specific IgG are

secreted by activated B-cells upon direct antigen recognition by surface expressed

IgG-receptors in combination with cytokines secreted by activated T-cells [64],[65].

Upon binding of opsonised particles by several FcγR, their cytosolic immunoreceptor

tyrosine-based motifs (ITAM) trigger particle uptake, accelerate phagosome

maturation and signal for the production of pro-inflammatory cytokine secretion [63].

1.3.4 The cell biology of phagocytosis in professional phagocytes

Phagocytosis of particles such as pathogens is the exclusive effector mechanism of

macrophages, neutrophils and DCs. As explained in detail above, phagocytic

receptors tether bacteria as M. tuberculosis to the phagosomal membrane and initiate

uptake. Overall the process of phagocytosis can be divided into three main steps: (i)

particle recognition, (ii) particle internalization and (iii) maturation of the particle-

containing phagosome to the phago-lysosome. Several PRRs engage in the process

of particle binding and thus multiple signaling cascades are triggered concomitantly.

Introduction

19

Herein, the process of FcγR-mediated phagocytosis is representatively explained in

greater detail since it is the best understood model of phagocytosis.

In order to trigger phagocytosis, FcγR have to sense and bind their ligand. Onwards

several receptors must cluster to elicit cellular responses and particle uptake [64]. This

spatial convergence brings together the cytosolic ITAM domains of the receptors.

Subsequently, Src-family kinases (SFK) phosphorylate the ITAM motifs.

Figure 1.4: Signalling events leading to actin polymerization during FcγR–mediated phagocytosis. Modified from [36]. See text for details.

The spleen tyrosine kinase (Syk) is able to bind the double phosphorylated ITAM-motif

and recruits several additional signaling proteins [65]: First, the linker of activated T

cells (LAT) is phosphorylated by Syk inducing docking of Grb2 and in turn Gab2 [66].

Additionally Gab2 is recruited to the receptor-complex by its interaction with

phosphatidylinositol-3,4,5-triphosphat (PI(3,4,5)P3), which is an important membrane-

lipid in the cytoplasmic lipid bilayer leaflet around receptor clusters. Phosphorylation

of Syk also recruits the CrkII adaptor proteins to the phagocytic cup important for

downstream signaling of FcγR [67].

At this time point after particle binding, lipids have an important role in orchestrating

phagocytosis. On the inner leaflet of the phagosomal membrane, phosphatidylinositol-

4,5-bisphosphat (PI(4,5)P2) is involved in pseudopod generation to form the

phagocytic cup [68]. Together with other anionic phospholipids such as

Introduction

20

phosphatidylserine (PS) and PI(3,4)P2, it creates a negative charge at the inner leaflet

of the plasma membrane. This attracts phosphatidyl-kinases such as PI4P-5K which

is essential for the lipid-homeostasis. Shortly after the transient synthesis of PI(4,5)P2,

it disappears allowing particle internalization probably by actin disassembly [69]. The

decrease of PI(4,5)P2 is mediated by PI-specific phospholipase Cγ and PI-3-kinase,

which are recruited to the phagocytic cup and hydrolyze PI(4,5)P2 to diacylglycerol

(DAG) or phosphorylate it to PI(3,4,5)P3 respectively [71],[73]. To further support

phagocytic cup formation, in addition phospholipase D is attracted to hydrolyze

phosphatidylcholine (PC) to phosphatidic acid (PA). With its cone shape, PA promotes

curvature of the membrane.

The formation of pseudopods and the phagocytic cup depend on the correct

orchestration of actin assembly and disassembly. This is mediated by small GTPases

of the Rho family. During phagocytosis these comprise Cdc42 and Rac1/2 which

stimulate formation of filopodia and lamellopodia, respectively [71]. Both cellular

protrusions are important for the formation of the phagocytic cup and the nascent

phagosome. Additionally the GTPase ARF6 delivers endosome membranes to

nascent phagosomes. Downstream of these GTPases, the assembly and disassembly

of (branched) actin filaments at the phagocytic cup is controlled by actin binding

proteins (APBs) and nucleation-promoting-factors (NPFs). Here the Arp2/3 complex

and the NPFs Wiskott-Aldrich syndrome proteins (WASP)/N-WASP or Scar/WAVE-

family proteins have an important role. Together with PI(4,5)P2, Cdc42 activates the

NPFs, which in turn activate Arp2/3 to promote actin assembly [72]. Furthermore,

several proteins such as myosins interact with actin filaments during phagocytosis. It

has been shown that myosin II, IXb and IC are important for particle engulfment and

that myosin X has a role in phagosome formation [73].

Introduction

21

Figure 1.5: Stages of phagosome maturation. Modified from [36]. After sealing of the nascent phagosome, maturation starts immediately. Through highly orchestrated fusion and fission events with early (EE), intermediate (IE), late (LE) and lysosomes (LY), the phago-lysosome matures. The phago-lysosome is a hostile organelle with a low pH of 4.5 containing hydrolytic enzymes as cathepsins, AMPs and reactive oxygen- and reactive nitrogen-intermediates. Specific phago-lysosomal markers are LAMP1 and LAMP2, high contents of the v-H+-ATPase and the absence of early endosome markers such as Rab5 or EEA1. Phagosomes of M. tuberculosis (Mtb) pause at an early phagosomal stage.

Introduction

22

After scission from the plasma membrane, the nascent phagosome interacts with early

endosomes thereby initiating phagosome maturation. These membrane fusion events

are mediated by Rab GTPases. Vps34 is targeted to Rab5-positive membranes via

Vps15 and catalyzes the conversion of PI to PI(3)P which is essential for progression

to the late phagosomal stage [74]. PI(3)P together with Rab5 recruit EEA1. The latter

has a fusion-promoting function as it interacts with syntaxin13, a soluble N-

ethylmaleimide-sensitive factor attachment protein (SNARE). SNARE-proteins are

universal mediators of membrane fusion. They form complexes composed of R-

SNARE (localized at donor membranes as early endosomes) and Q-SNARE (localized

at acceptor membranes as early phagosomes) proteins. These hairpin-like protein

complexes bring donor- and acceptor membranes in close proximity, thereby reducing

the free energy barrier for membrane fusion [36].

Despite fusion with early endosomes, the volume of the early phagosome remains

constant. This is due to the fission of endosomes recycling from phagosomes to e.g.

retrieve cell-surface proteins as the transferrin-receptor (TfR) back to the plasma

membrane. Endosomal fission from phagosomes is mediated by an elaborate network

of signaling, budding and tubulating components as Rab4, Rab11, Eps15, the hetero-

oligomeric complex (COPI) and the retromer complex [36]. Another mechanism

maintaining phagosome size is the formation of intraluminal vesicles (ILVs). At the

intermediary phagosomal stage ILVs develop via invagination and pinching of the

phagosomal membrane with the help of the endosomal sorting complex for transport

(ESCRT) machinery and PI(3)P binding proteins Hrs and SNX3 [75].

With ongoing fusion and fission of the phagosome with intermediary and late

endosomes, phagosome maturation proceeds. At the late stage, the phagosome

differs significantly from its early stage. It loses early markers such as Rab5, gains late

markers like Rab7, acidifies and acquires acid hydrolases. The exchange between

Rab5 and Rab7 is an essential step towards maturation. Simultaneously, late

phagosomes acquire the lysosome-associated membrane proteins 1 and 2

(LAMP1/2). Apart from providing membrane integrity and protection against

membrane-active enzymes, LAMP1 and 2 were recently shown to be important for

phagosome maturation by recruiting Rab7. Another characteristic of late phagosomes

is the presence of the unique lipid lysobisphosphatidic acid (LBPA) inside ILVs.

Furthermore, acidification of the maturing phagosome is a consequence of the

constant accumulation of proton (H+) pumps as the vacuolar ATPase (v-H+-ATPase).

Introduction

23

This multimeric protein complex translocates H+ across the phagosomal membrane at

the expense of ATP lowering the early pH of 6.3 to 4.5 [36].

Finally, the late phagosome fuses with lysosomes to form phago-lysosomes. This final

fusion event is, in part, coordinated by the formation of a specific SNARE complex

made of syntaxin7 and VAMP7. The phago-lysosome is the intracellular compartment

that is specifically designed to digest proteins, lipid-membranes, carbohydrates and

thus also microbes [36]. The v-H+-ATPase-mediated acidification of the late

phagosome starts a well-defined process of activation of microbicidal effectors. Due

to the low pH, microbial growth is inhibited, the enzymatic activity of proteases such

as cathepsin B, D and L is optimized at pH of 4.5 and the natural-resistance-associated

macrophage protein1 (NRAMP) is recruited. NRAMP is able to export metal ions such

as iron (Fe3+) from the phago-lysosome to sequestrate ions essential for growth of

intra-phagosomal bacteria [76]. The phago-lysosome harbors several bactericidal

peptides and proteins: lysozyme is able to hydrolyze peptidoglycan; the main structural

components of bacterial cell-walls. Furthermore, cationic antimicrobial peptides

(cAMPs) interact with anionic bacterial membranes and generate pores allowing the

diffusion of ions across the cell-wall. With high proton concentrations and the activation

by pro-inflammatory cytokines as IFN-γ, the macrophage is also capable of producing

reactive oxygen intermediates (ROI) by the NADPH oxidase (NOX2) and reactive

nitrogen intermediates (RNI) by nitrous oxide synthase 2 (iNOS) (Figure 1.6). ROI and

RNI contribute to the elimination of pathogens in phago-lysosomes by damaging

proteins, lipids and DNA/RNA.

Figure 1.6: Production of ROI and RNI in the phagosome. Modified from [77]. The membrane-standing proteins iNOS and NOX2 catalyse the production of NO-and O2-radicals from oxygen. These highly reactive compounds further react to form peroxinitrite (ONOO-) or nitrogen dioxide radicals (NO2

-) and hydrogen peroxide (H2O2), respectively. All compounds are able to damage proteins, lipids, DNA and RNA.

Introduction

24

1.4 Host-pathogen interactions in the phagosome

As outlined, M. tuberculosis infects the host by engaging phagocytic receptors to enter

professional phagocytes as macrophages to abuse as host-cells. After invasion, the

pathogen ensures survival and colonization by deviating the microbicidal mechanism

of phagosome maturation. For that purpose, M. tuberculosis expresses an array of

virulence factors. The main reservoir of M. tuberculosis virulence factors is their unique

and highly complex cell-wall. Here lipids and proteins synergize to disturb the

macrophage microbicidal properties. This chapter gives a detailed overview about the

secreted virulence factors as well as the components of the mycobacterial cell-wall

and, as far as known, their molecular function in virulence.

Per definition, virulence factors are peptides, proteins or lipids of pathogens, whose

inactivation leads to a significant loss in pathogenicity or virulence but fails to impair

the bacterial growth in standard growing media [77]. This criterion comprises a very

large spectrum of candidates including the genes and proteins required for expression,

transport and positioning of virulence factors. In the present work, the focus lies on

lipids that are at the molecular forefront of direct interaction with the host-cell.

The reference laboratory strain M. tuberculosis H37Rv harbors 14 regions of difference

in its genome (RD 1-14) which are absent in the vaccine strain M. bovis BCG and are

thought to be related to pathogenicity. Together with 6 regions termed H37Rv deletion

1-5 (RvD1-5) and the M. tuberculosis specific deletion (TbD1), these regions are

believed to code for the virulence factors of the MTBC. However, the virulence-

associated genes of M. tuberculosis are not classically concentrated on pathogenicity

islands as in other bacteria such as Salmonella but rather are widely distributed

throughout the genome.

1.4.1 The role of cell envelope and secreted proteins in M. tuberculosis

virulence

Proteomic studies of the mycobacterial cell envelope revealed more than 500 proteins

[77]. Most of them are thought to be important for cell-wall homeostasis but 5 % might

have a role in virulence. Cell-wall proteins include the outer membrane proteins

(OMPs) localized in the mycobacterial outer membrane (MOM), cell-wall-associated

or secreted lipo- and glycoproteins.

The delivery of virulence proteins across the cell envelope during infection of host-

cells is mediated via specialized type 1-4 secretion systems (T1-4SS) [78]. The

Introduction

25

genome of M. tuberculosis encodes for at least four types of secretion systems but

only T2SS and the Mycobacteria specific T7SS have a role in virulence. The ESAT6

secretion system 1 (ESX-1) is a specialized T7SS for secretion of virulence relevant

proteins ESAT6 (ESXA), CPF10 (ESXB), EspA-D and EspR. ESAT6 and CFP10,

encoded on RD1 and indispensable for virulence of M. tuberculosis, are small proteins

of 9 kDa and 10 kDa respectively, which form heterodimers [78]. Their virulence

function has been linked to M. tuberculosis escape from the phagosome into the

cytoplasm and spread to uninfected cells as well as inhibition of apoptosis [79].

OMPs are MOM-associated and therefore can directly interact with the host-cell [77].

Lipo- and glycoproteins are exported into the cell-wall or the host-cell cytosol via T2SS

or general Sec secretory pathways. The fibronectin binding proteins are a complex of

three proteins (FbpA-C), better known as the antigen 85 complex (Ag85). Besides

being a mycolic acid transferase, this complex is the major secreted protein of

Mycobacteria and has been shown to mediate the adhesion of the pathogen to

fibronectin on mucosal surfaces, thereby facilitating entry to the host. The six Mce

(mammalian cell entry) proteins are secreted or surface exposed. It has been shown

that Mce1 supports entry of mycobacterial pathogens into mammalian cells and

survival inside macrophages [80]. Adhesion of M. tuberculosis to epithelial cells is also

accomplished via HbhA (heparin-binding protein). HbhA tethers Mycobacteria to

epithelial cells and promotes bacterial aggregation and primary biofilm formation [81].

Furthermore, the 15 kDa lipoprotein was shown to interact with TLR2 possibly

regulating immune responses in favor of mycobacterial survival in the phagosome.

The 27 kDa lipoprotein AG P27 (LprG) has been shown to have a role in infection by

suppression of the host immune response and to bind DC-SIGN mediating adhesion

[77].

M. tuberculosis harbours also three cell-wall associated secretory glycoproteins: the

MPT83 antigen, the 45-47-kDa alanine-proline-rich antigen (Apa, Rv1860) and LqpH

(19 kDa antigen). All three are believed to be potential adhesins and thus have role in

host-cell attachment. Additionally LqpH binds MR, is able to inhibit antigen

presentation of MHC class II molecules in a TLR2-dependent manner and induces

apoptosis for the cell to cell spread of the pathogen [82]. Furthermore, Psts-1 (38 kDa

glycoprotein) was also shown be secreted and to interact with TLR2 and TLR4

resulting in induction pro-inflammatory cytokines [77].

Introduction

26

Pathogens of the MTBC harbor several secreted virulence proteins which increase the

resistance to host toxic compounds as ROI and RNI, arrest phagosome maturation

and prevent apoptosis. M. tuberculosis employs at least six proteins that are secreted

into the phagosomal lumen via SecA2 to directly interfering with ROI and RNI. SodC,

KatG, AhpC, TpX, Mel2 and putatively Acr2 have superoxide dismutase activity that

either inhibits production of O2 radicals or detoxifies H2O2. Further, M. tuberculosis

possesses several proteins suggested to be involved in inhibition of phagosome

maturation. These include Ndk, PtpA and PE_PGRS30. Ndk was shown to inhibit

recruitment of Rab7-GTP and Rab5-GTP to phagosomes and inactivate their function

by dephosphorylation [77]. Furthermore the phosphatase PtpA dephosphorylates

VPS33B, a host protein involved in regulation of membrane fusion. PtpA also binds to

the v-H+-ATPase machinery, thereby inhibiting luminal acidification. The detailed role

of PE_PGRS30 is unknown, although its deletion renders mutants unable to inhibit to

phago-lysosome fusion [77]. Recently the secreted acid phosphatase SapM was found

to dephosphorylate PIP(3)P, that has an essential role in phagosome maturation [83].

Inhibition of apoptosis is another strategy to survive inside professional phagocytes

since the programmed cell death is one of the major mechanisms of the innate immune

response to contain the spread of pathogens. Virulent Mycobacteria modulate host-

cell death by switching from apoptosis to necrosis. This is achieved by controlling the

production of ROI and RNI-mediated apoptosis by NuoG, SecA2/SodA, Rv3600-3653c

and protein kinase PknE.

1.4.2 The role of cell envelope lipids in M. tuberculosis virulence

The mycobacterial cell envelope is composed of three layers: (i) the outermost layer

composed of the capsule and the mycobacterial outer membrane (MOM), (ii) the cell-

wall core composed of arabinogalactan (AG) covalently linked to peptidoglycan (PG)

and (iii) the plasma membrane (PM) (Figure 1.7). The mycobacterial cell-wall

contributes to virulence also by being highly impermeable impeding entry of toxic

molecules as antibiotics.

Introduction

27

Figure 1.7: Schematic representation of the composition of the mycobacterial cell-wall. Modified from [84]. The mycobacterial cell-wall is partitioned in three layers. The capsule constitutes the outermost surface, followed by the mycobacterial outer membrane (MOM) or mycolic acid layer. Peptidoglycan (PG) and arabinogalactan (AG) build the core of the cell-wall. The plasma membrane or cell membrane (CM) surrounds the lumen of the pathogen.

(i) The plasma membrane (PM) is an asymmetric bilayer composed of phospholipids

as phosphatidylglycerol (PG), P2G, phosphatidylethanolamine (PE) and proteins.

Mannosylated lipoarabinomannan (ManLAM) and phosphatidyl-inositol mannosides

(PIMs) are anchored here via their phosphatidyl-myo-inositol residue.

(ii) The cell-wall core is composed of arabinogalactan (AG) and peptidoglycan (PG).

AG is a polymer made of arabinose and galactose monosaccharides that tethers the

mycolic acid layer to peptidoglycan (PG). The PG polymer is orientated orthogonal to

the plasma membrane and consists of sugars as N-acetyl-α-D-glucosamine and

modified muramic acid and the amino acids L-alanyl-D-isoglutaminyl-meso-

diaminopimelyl-D-alanine (L-ala-D-glu-A2pm-D-Ala) similar to Gram-positive and

Gram-negative bacteria. The peptide chain can be cross linked to the peptide chain of

another strand forming the 3D mesh-like layer [23]. The cell-wall core forms a rigid

layer outside the PM providing cellular shape and strength as well as scaffold for AG

and the MOM.

(iii) The mycobacterial outer membrane (MOM) is composed of a bilayer membrane of

mycolic acids, mannosylated molecules, acyltrehaloses, mycocerate-containing

glycolipids and glycoproteins.

The mannosylated molecules comprise glycoproteins and (lipo) glycans as PIMs,

lipomannan (LM), lipoarabinomannan (LAM) and mannose-capped LAM (ManLAM).

These molecules are non covalently linked to the mycolic acids of the MOM (PIM) or

http://en.wikipedia.org/wiki/Arabinose

http://en.wikipedia.org/wiki/Galactose

http://en.wikipedia.org/wiki/Sugars

Introduction

28

the phospholipids of the plasma membrane (LM and LAM) through hydrophobic

interactions or their phosphatidyl-myo-inositol part [85].

PIMs can be divided into two groups concerning their number of mannoses: the lower

(PIM2-4) and higher order PIMs (PIM5-6). PIMs exhibit virulence function via the

induction of phagocytosis through binding to CR3 (lower) and the limiting of

phagosome-lysosome fusion events by binding to MR (higher). Furthermore, PIMs

facilitate early endosomal fusion in a Rab5-dependent manner [86]. The most

abundant forms of PIM in Mycobacteria are di-, tri- or tetra-acylated PIMs. LMs engage

with DC-SIGN. As heterodimer with TLR2/TLR1, DC-SIGN then induces apoptosis and

has pro-inflammatory effects on host-cells [82]. Mannose-capped LAM (ManLAM) is

one of the most abundant mannosylated lipids in the cell-wall of slow-growing

pathogenic Mycobacteria. Its influence on host-cell functions is diverse. Upon contact

between the pathogen and the host-cell, ManLAM interacts through its mannose

residues with the MR on macrophages and induces phagocytosis. By engaging MR,

the early phagosomal state of the pathogen-containing vacuole is maintained. Via

binding to DC-SIGN, phagocytosis is triggered as well and anti-inflammatory pathways

are up-regulated [87]. ManLAM was also shown to intercalate into the phagosomal

membrane via its PI-anchor disturbing lipid microdomains. Furthermore, it interferes

with phagosomal maturation by interfering with the recruitment of PI(3)P and Rab5 to

the phagosomal membrane [87]. Inside the phagosome it reduces phagosomal

microbicidal activity by inhibiting production of ROI, RNI and inflammatory cytokines

and apoptosis. ManLAM is also shedded from the MOM and has been shown to traffic

inside macrophage membranes [88].

The acyltrehaloses comprise the sulfatides (SL), diacyltrehaloses (DAT),

triacyltrehaloses (TAT), polyacyltrehaloses (PAT), trehalose monomycolate (TMM)

and trehalose dimycolate (TDM).

Figure 1.8: Acyltrehaloses TMM and TDM of the mycobacterial outer membrane. Modified from [82]. TMM and TDM are made of a headgroup of the sugar trehalose that can be esterified at position 6 and/or 6´ to one or two mycolic acids, respectively.

Introduction

29

The exact function of SL in mycobacterial virulence, in particular in intracellular

trafficking of Mycobacteria, remains to be fully understood. However, DAT, TAT, PAT

lipids, although non-essential for mycobacterial viability in vitro, probably contribute to

the properties of the cell surface and the permeability barrier formed by the cell

envelope [77].

TDM, the most abundant lipid in the mycobacterial cell-wall, is composed of trehalose

esterified with one (TMM, precursor of TDM) or two (TDM) mycolic acids at position 6

and/or 6’ (Trehalose-6,6-dimycolate) (Figure 1.8). Mycolic acids are long-chain fatty

acids, α-branched and β-hydroxylated with different chain lengths (from 60 to 90

carbon atoms in Mycobacteria) and chemical functional groups, such as double bonds,

cyclopropanes or oxygenated functions located at two defined positions of the

meromycolic chain [87]. TDM is also referred to as “cord factor” since its presence

alters the colony morphology to rope-like forms [23]. Alike ManLAM, TDM has various

functions relevant for M. tuberculosis early in infection as well as in granuloma

formation [89]. TDM has been proposed to be the main glycolipid interfering with

phagosome maturation. In 2003, Indrigo et al. showed that TDM restores virulence

function of delipidated Mycobacteria [90]. Moreover beads coated with TDM are able

to delay phagosome maturation and retain a close proximity to the phagosomal

membrane, prerequisite to inhibit phagosome maturation [91]. However, in IFN-γ-

activated macrophages, the inhibitory effect of TDM on phagosome maturation is

abolished by NO [91]. Furthermore, TDM alone is sufficient to induce granuloma

formation when intravenously injected on oil droplet formulations into mice. TDM-

mediated granuloma formation is mediated by Mincle. Engagement of Mincle activates

the Syk-Card9 signalling pathway in macrophages, which is required for activation of

macrophages in vitro and for granuloma formation in vivo following injection [92].

However, the exact molecular function of TDM remains unknown.

The mycerosate-containing lipids are phtiocerol dimycerosates (PDIM) and phenolic

glycolipids (PGL). Besides the importance of PDIM in multiplication of M. tuberculosis,

the lipid has been shown to inhibit secretion of pro-inflammatory cytokines and was

proposed to inhibit phagosome maturation by incorporating into host membranes

disturbing their organization [36],[93]. Also, cell surface associated PDIM were shown

to mask underlying PAMPs avoiding activation of pro-inflammatory processes [94].

PGL from M. marinum has been found to inhibit maturation of bead phagosomes [95].

Introduction

30

(iv) The capsule is a loosely-bound, non-covalently linked structure on the outermost

compartment of the cell. In M. tuberculosis it is composed of proteins, such as porins

for nutrient uptake, oligosaccharides and only small amounts of lipids such as PIMs

[82]. Capsular components are prone to release upon contact with the host-cell or

within the phagosome. The oligosaccharides comprise α-D-glucan, D-arabino-D-

mannan and D-mannan and were described to mediate phagocytosis via non-opsonic

binding to CR3 [82].

1.5 Objectives

The aim of this PhD-thesis was to investigate how TDM exerts its virulence function

inside the phagosome of macrophages. We hypothesize that TDM manipulates host-

cell biology through interaction with targets at the phagosome interface, in order to

inhibit phagosome maturation. To identify putative interaction partners, the lipid-coated

bead model should be used. Therefore, the following aims had to be accomplished:

i. establishment of a protocol for isolation and purification of magnetic lipid-

coated bead phagosomes from macrophages suitable for mass spectrometry

based proteomic and lipidomic analysis,

ii. analysis of control and TDM bead phagosomes proteomes and lipidomes to

identify potential host-cell-derived direct or indirect interaction partners,

iii. evaluation of selected potential host-cell targets of TDM for their role in TDM-

and M. tuberculosis-mediated inhibition of phagosome maturation and

intracellular survival.

Material and Methods

31

2 Material and Methods

2.1 Material

2.1.1 Consumables

Table 2-1: Consumables

Designation Manufacturer

75 cm2 flask Corning

5 ml screw top vial Chromacol

Dynabeads M-280 tosylactivated Life Technologies

50 ml tube, blue screw cap Greiner Bio-One

15 ml centrifuge tubes Corning

Filtropur S plus 0.2 Sarstedt

1000 µl pipette tips Sarstedt

200 µl pipette tips Sarstedt

10 µl pipette tips Molecular Bio Products

25 ml stripette Costar Corning




25 cm cell scraper Sarstedt

Syringe, plastipak 1 ml Becton Dickinson

Canula, microlance 3, 23 G1, Nr. 16 Becton Dickinson

FACS tube, 5 ml Becton Dickinson

1.5 ml, 2 ml reaction-tube Sarstedt

96-well plate, flat bottom Corning

2 ml protein lobind tube Eppendorf


4-10 % criterion-TGX-precast gel Biorad

LC-vial 100 µl Chromacol

Coverslips, 10 mm, round VWR

Canula, microlance 3, 37G 3/4, Nr. 20 Becton Dickinson

Parafilm laboratory film BEMIS


IPFL00010, immobilon-FL PVDF, 0.45 µm Merck

Petri dish Sarstedt

http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430052%28Lifesciences%29&categoryname=General+Labware+and+Equipment%28Lifesciences%29%7cCentrifuge+Tubes+%28Lifesciences%29%7cCentrifuge+Tubes%2c+Disposable+Plastic+%28Lifesciences%29%7c15mL+Screw+Cap+Conical+Bottom+Centrifuge+Tubes%28Lifesciences%29


32

Glass pasteur pipettes Brand

1.5 ml glass vials Marchery Nagel

2 ml eppendorf reaction tubes Eppendorf

HPLC vial with insert, 0.3 ml Chromacol

Caps for HPLC vials Chromacol

2.1.2 Chemicals

Table 2-2: Chemicals


Dulbecco’s Modified Eagle’s Medium (DMEM) PAN Biotech

Fetal bovine serum (FBS/FCS) Merck

L-Glutamine PAN Biotech

Penicillin/Streptomycin PAN Biotech

Trypan blue solution 0.4 % Sigma-Aldrich

Trichlormethan/Chloroform Carl Roth

Phosphate buffered saline (PBS) PAN Biotech

BSA (albumin bovine fraction V) Serva

D(+)-Saccharose Carl Roth

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid

(HEPES)

Sigma-Aldrich

1,4-Dithiothreit Carl Roth

Gelatin from porcine skin for electrophoresis, type A Sigma-Aldrich

Complete EDTA free, protease inhibitor cocktail

tablets

Roche

Deoxyribonuclease I from bovine pancreas type IV Sigma-Aldrich

Trypsin gold, mass spectrometry grade Promega

Ficoll PM 70, type 70 Sigma-Aldrich

Roti-load Carl Roth

StrataClean resin Agilent Technologies

Hydrochloric acid fuming 37 % Merck

Deionized water, millipore water Merck

2-Nitrophenyl β-D-galactopyranoside (ONPG) Sigma-Aldrich

Triton X 100 Carl Roth

Citric acid, anhydrous Sigma-Aldrich

Sodium citrate monobasic Sigma-Aldrich

Sodium carbonate Carl Roth


33

Ammonium persulfate Sigma-Aldrich

Potassium thiocyanate (KSCN) Sigma-Aldrich

Iron (III) chloride (FeCl3) ABCR

Trizma base Sigma-Aldrich

Glycerol Sigma-Aldrich

Sodium-dodecyl sulphate (SDS) Carl Roth

2-Mercaptoethanol Sigma-Aldrich

Bromphenol blue–xylene cyanole dye solution Sigma-Aldrich

Acetic acid, 100 % Carl Roth

Ethanol Carl Roth

Colloidal coomassie Sigma-Aldrich

Ammonium bicarbonate Fluka

Acetonitril Carl Roth

Aeris C18 reversed-phase material Phenomenex

Paraformaldehyde Carl Roth

Middlebrook 7H9 broth Beckton Dickinson

Oleic albumin dextrose catalase (OADC) Sigma-Aldrich

Hygromycin B Sigma-Aldrich

LatrunkulinA Sigma-Aldrich

Dimethyl sulfoxide Sigma-Aldrich

Ammonium chloride Carl Roth

Goat serum PAN Biotech

4',6-Diamidino-2-phenylindole dilactate (DAPI) Life Technologies

Confocal matrix Micro-Tech-Lab

Cell line nucleofector kit V Lonza

Pierce 660 nm protein assay reagent Life Technologies

Tris base Sigma-Aldrich

30 % Acrylamide/Bisacrylamide solution Biorad

Sodium dodecyl sulphate Sigma-Aldrich

Tetramethylethylenediamine (TEMED) Biorad

Glycine AMRESCO

Methanol VWR

Powdered milk Carl Roth

Tween 20 Sigma-Aldrich

Tween 80 Carl Roth


34

ECL Western blotting detection reagent GE Healthcare

Mycobacteria 7H11 Agar Beckton Dickinson

Cattle serum PAN Biotech

Methyl tert-buthyl ether for HPLC (MTBE) Sigma-Aldrich

Water LC-MS Sigma-Aldrich

Methanol LC-MS Sigma-Aldrich

Acetyl chloride Sigma-Aldrich

Chloroform for HPLC Sigma-Aldrich

Ammonium hydroxide solution Sigma-Aldrich

Ammonium acetate for mass spectrometry Sigma-Aldrich

2-Propanol LC-MS Sigma-Aldrich

2.1.3 SiRNA

Table 2-3: siRNA


ON-TARGETplus SMARTpool – mouse-annexinA1 GE Healthcare

ON-TARGETplus SMARTpool – mouse-annexinA6 GE Healthcare

ON-TARGETplus SMARTpool – mouse-cofilin1 GE Healthcare

ON-TARGETplus SMARTpool – mouse-profilin1 GE Healthcare

ON-TARGETplus SMARTpool – mouse-SNAP23 GE Healthcare

ON-TARGETplus SMARTpool – mouse-VAMP3 GE Healthcare

ON-TARGETplus SMARTpool – mouse-WASH1 GE Healthcare

ON-TARGETplus SMARTpool – mouse-NTR GE Healthcare

2.1.4 Lipids

Table 2-4: Lipids


Trehalose-6,6'-dimycolate from M. bovis (BCG) BioClot

17:0-14:1, PC Avanti Polar Lipids

17:0-20:4, PC Avanti Polar Lipids

17:0-14:1, PE Avanti Polar Lipids

17:0-14:1, PG Avanti Polar Lipids

17:0-14:1, PS Avanti Polar Lipids

D18:1-12:0, Sphingomyelin Avanti Polar Lipids

C25 Ceramide Avanti Polar Lipids

17:0, Lyso-PC Avanti Polar Lipids


35

16:0-17:0, BMP (R,R) Avanti Polar Lipids

2.1.5 Antibodies and dyes

Table 2-5: Antibodies and dyes


Anti-annexin A1 Abcam

Anti-annexin VI Abcam

Anti-cofilin1 Abcam

Anti-profilin1 Abcam

Anti-SNAP23 Acris Antibodies

Anti-cellubrevin (VAMP3) Abcam

Anti-WASH1 Atlas Antibodies

LAMP1 1D4B self made

Anti-rat cy3 Life Technologies

Anti-rabbit cy2 Life Technologies

Anti-rabbit cy5 Life Technologies

Anti-rabbit alexa405 Life Technologies

Anti-rat HRP Jackson Immuno Research

Anti-rabbit HRP Jackson Immuno Research

Lysotracker red Life Technologies

PhalloidinAlexa488 Life Technologies

PhalloidinAlexa594 Life Technologies

2.1.6 Cell-lines

Table 2-6: Cell-lines


RAW264.7 macrophage cell line ATCC, US

2.1.7 Bacteria

Table 2-7: Bacteria

Designation Source

M. tuberculosis H37Rv-GFP Tanya Parish [96]

2.1.8 Hardware

Table 2-8: Hardware

Type Manufacturer

CO2 cell Incubator, HERA-cell 240 Thermo Fisher Scientific


36

DynaMag-2 magnet Life Technologies

Tube rotator SB3 Stuart

HERAEUS multifuge 3SR+ Thermo Fisher Scientific

Rotor SORVALL HERAEUS 75006445 Thermo Fisher Scientific

Ultrasonic bath, sonorex super RK255H Bandelin

Clean bench, MSC-advantage class II biological

Safety cabinets

Thermo Fisher Scientific

Finnpipette, 1-10 μl, 10-100 μl, 100-1000 μl Thermo Fisher Scientific

Pipetboy, finnipette Thermo Fisher Scientific

Metal douncer WHEATON

1.5 ml tube heater, univortemp MT100 Universal Labortechnik

TCS SP5 confocal microscope Leica

Nikon eclipse TS100 Nikon GmbH

Table-top centrifuge, HERAEUS PICO 17 Thermo Fisher Scientific

Table-top centrifuge, HERAEUS FRESCO 17 Thermo Fisher Scientific

Table-top centrifuge 5424R Eppendorf

Vacuum-centrifuge for speed vacuum ScanVAC

Tecan sunrise microplate reader Tecan Group Ltd.

FACSAria IIU Becton Dickinson

Biorad power pac HC Biorad

Mini-PROTEAN tetra handcast systems Biorad

EASY-nLC II system Thermo Fisher Scientific

Proxeon 1000 system Thermo Fisher Scientific

P-2000 laser puller Sutter Instrument

Sorcerer-SEQUEST SageN Research

LTQ orbitrap velos Thermo Fisher Scientific

UV/Vis spectrophotometer Jenway

Neubauer-improved counting chamber Paul Marienfeld

Nucleofector 2b device Lonza

Tecan infinite M200 microplate reader Tecan Group Ltd.

Mini trans-blot cell Biorad

Horizontal shaker Heidolph Instruments

Amersham hypercassette autoradiography GE Healthcare

Amersham ECL Western blotting detection

reagent

GE Healthcare

http://www.bio-rad.com/de-de/product/electrophoresis-chambers/mini-protean-tetra-handcast-systems?pcp_loc=catprod

http://www.bio-rad.com/de-de/product/mini-trans-blot-cell?pcp_loc=catprod


37

Medical film processor Konica Minolta

GS-800 calibrated densitometer Biorad

1100 HPLC Agilent Technologies

BETASIL diol-100 column ThermoFisher Scientific

Apollo dual ESI/MALDI ion source Bruker Daltonics

Bruker apex Qe FT-MS Bruker Daltonics

Q-TOF ultima, equipped with ESI source Waters

2.1.9 Software

Table 2-9: Software


Microsoft office 2010 Microsoft

Magellan 6.0/7.0 Tecan Group Ltd.

Scaffold 4 Proteome Software

Compass apex control 3.0.0 Bruker Daltonics

HyStar 3.2 Bruker Daltonics

Data analysis 3.2/4.0 Bruker Daltonics

LipidXplorer [97]

MassLynx 4.0 Waters

Graph pad prism 5.0/6.0 Graph Pad Software


38

2.2 Methods

2.2.1 Isolation and purification of lipid coated bead phagosomes from

macrophages for mass spectrometry analysis

2.2.1.1 Cell strains and culture conditions

RAW264.7 macrophages were cultivated in Dulbecco's Modified Eagle's Medium

(DMEM)-media supplemented with 10 % (v/v) fetal calf sera (FCS), 1 % (v/v) L-

glutamine and 1 % (v/v) penicillin/streptomycin at 37 °C and 7.5 % CO2. The media is

further referred to as D10-media. For infection experiments with M. tuberculosis GFP,

D10-media without antibiotics was used.

2.2.1.2 Cell counting

RAW264.7 macrophages were washed with 5 ml PBS, scratched into 5 ml PBS and

the cell concentration was determined using a Neubauer counting chamber. Therefore,

10 µl of the cell suspension was pipetted in a Neubauer counting chamber and cells

were counted using a light microscope. Live/dead staining was accomplished using

Trypan blue.

2.2.1.3 Coating of beads with M. bovis BCG cell-wall lipid Trehalose-6,6’-

dimycolate

Trehalose-6,6-dimycolate (TDM) was solved in chloroform to a concentration of

5 mg/ml, filled to a 5 ml glass-vial and stored at -20 °C until further use. Tosylactivated

Dynabeads M-280 were stored at 4 °C up to one year.

The number of control and TDM coated beads was adjusted to the number of T75-

flasks prepared with a confluent monolayer of RAW264.7 macrophages. A confluent

monolayer of RAW264.7 macrophages usually contains 107 cells. “Bead infection” was

carried out with a multiplicity of infection (MOI) of 10. Likewise, per 107 macrophages,

108 beads were prepared.

Required amounts of beads were collected, the storage solution was removed using

the DynaMag-2 magnet and beads were washed thrice with 1 ml PBS using the

DynaMg-2 magnet. Then 1 % bovine serum albumin (BSA)-solution was prepared in

20 ml PBS and sterilized by filtration through a 0.20 µm filter. Subsequently, beads

were incubated in sterile 1 % BSA in PBS for 3 h at room-temperature (RT) on a tube

rotator. Beads were centrifuged for 5 min at 1500 rpm, the supernatant was discarded

and beads were solved in 1 ml PBS. Again, beads were washed thrice with 1 ml PBS


39

using the DynaMg-2 magnet. For labeling BSA-coated beads with TDM, 50 µg TDM

per 107 beads were used. Therefore, a distinct amount of TDM was placed into a glass-

vial and vaporized using gaseous nitrogen. Subsequently, beads solved in 1 ml PBS

were added and incubated for 1.5 h in an ultrasonic bath. Control beads (BSA-coated

only) were treated likewise but without TDM. Beads were ready to use directly or were

stored up to one week at 4 °C until further use.

2.2.1.4 “Infection” of macrophages with lipid coated beads

One day prior “bead infection” experiments, four T75-flasks were equipped with 107

RAW264.7 macrophages and incubated over-night (ON) in D10-media at 37 °C and

7.5 % CO2. Control and TDM coated beads were prepared as outlined in 2.2.1.3.

“Bead infection” with control and TDM beads was carried out in parallel.

The experiment was started by removing media from RAW264.7 macrophages grown

in T75-flasks and washing the macrophages with 5 ml PBS. Then 5 ml ice-cold D10-

media was added. Therefore, T75-flasks were put upright in order to avoid direct

contact between the media and the cells at this time point. Subsequently control and

TDM beads were pipetted directly into ice-cold D10-media and mixed carefully by

shaking. Bead suspension was poured onto the cells by putting flasks in a horizontal

position and gentle shaking. Macrophages were further incubated for 5 min at 4 °C

and 5 min at 37 °C and 7.5 % CO2. Beads not attached to the cells were removed by

discarding the D10-media and washing once with warm 5 ml D10-media. Then 10 ml

D10-media were added and cells were incubated at 37 °C and 7.5 % CO2.

2.2.1.5 Isolation and purification of lipid coated bead phagosomes from

macrophages

After desired time points, D10-medium was discarded and cells were washed with 5 ml

PBS. Subsequently, 10 ml ice-cold PBS were added and macrophages containing

bead phagosomes were scratched of the flask, transferred to a 50 ml falcon tube and

spun down 5 min at 1500 rpm at 4 °C. In case more than 1 T75-flask with 107

macrophages (control or TDM) was prepared, all technical replicates were pooled from

this step on. Supernatant was discarded and pelleted cells were solved in 1 ml ice-

cold homogenization buffer (HB, 250 mM sucrose, 20 mM 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES), 1 mM dithiothreitol (DTT), 0.1 % (v/v) gelatin,

1 tablet protease inhibitor) and transferred into the douncer. To disrupt the cells, the

stamp of the douncer was pushed down strongly 15-30 times and the destruction

progress was followed under the light-microscope. Subsequently, samples were


40

pushed through a syringe needle gauge (0.20 µm) to further lyse cells. Samples were

supplied with 50 u/ml DNase I and incubated for 10 min at 37 °C, before samples were

washed three times with 1 ml ice-cold HB and three times with ice-cold PBS by using

the DynaMag-2 magnet. For further purification and removal of cell-debris, samples

were briefly digested with 2.5 µg trypsin per 1 mg protein for 10 min at 37 °C. Samples

were washed three times with HB using the DynaMag-2 magnet. Samples were loaded

onto a 15 % Ficoll-gradient (1.5 g Ficoll-powder solved in 10 ml HB) and centrifuged

for 15 min at 2220 rpm at 4 °C. The supernatant was then carefully removed by

pipetting and the pellet was solved in 1 ml HB. The samples were again washed three

times with 1 ml ice-cold HB and three times with ice-cold PBS using the DynaMag-2

magnet. At this point, 100 µl of every sample were saved for further analysis with the

β-galactosidase assay (2.2.1.7.1) and the ferrozoin-assay (2.2.1.7.2). For lipidomic

analysis and Western blot, the purification process was stopped here and samples

were stored at -20 °C until further use.

2.2.1.6 Purification of lipid coated bead phagosomes via sorting by FACS

For further purification of bead phagosomes by sorting via FACS, samples were placed

in FACS tubes and 2 ml ice-cold PBS (final volume 3 ml) was added. Sorting by FACS

was carried out using a 70 µm nozzle size, with a low flow rate of 4 µl/sec resulting in

a sorting speed of approximately 4000-7000 events/sec. All together, 107 control and

TDM beads phagosomes were sorted into five collecting 15 ml falcon-tubes (5x 2x106

beads phagosomes per tube) prepared with 5 ml ice-cold PBS supplemented with

protease-inhibitors. During the sorting process, tubes were kept at 4 °C for the 6-

10 min sorting time. The sorting efficiency was evaluated by re-analyzing the sorted

bead phagosome fraction.

Control and TDM bead phagosomes were re-isolated from the sorting supernatant

using the DynaMag-2 magnet, solved in 12.5 µl sorting supernatant and stored at -

20 °C until further use. The sorting supernatant was pooled in a 50 ml falcon-tube and

remaining proteins were precipitated by using 20 µl “StrataClean beads” per sample.

For this purpose, StrataClean beads had to be prepared as follows: 20 µl were

incubated with 180 µl hydrochloric acid (HCl) for 6 h at 100 °C. StrataClean beads

were spun down 10 min at 10.000 xg, the HCl-supernatant was removed, StrataClean

beads were solved with 1 ml of sorted supernatant, added to the remaining sorted

supernatant and incubated ON at 4 °C on a tube-rotator. The next day, StrataClean

beads were spun down for 45 min at 10.000 xg, the supernatant was removed and the


41

pellet was solved in 1 ml deionized water. Again StrataClean beads were spun down

for 5 min at 20.000 xg, the supernatant was removed and samples were dried in a

vacuum centrifuge. Finally, StrataClean beads were solved in 12.5 µl of sorted

supernatant and stored at -20 °C until further use.

2.2.1.7 Quality control and quantification of lipid coated bead phagosomes

2.2.1.7.1 Lysosomal β-galactosidase assay

Validation of TDM-mediated inhibition of bead phagosome maturation was evaluated

by measuring lysosomal β-galactosidase activity.

The protocol was adapted from a previous report [98]. 150 µl of the lysosomal β-

galactosidase reaction mix (10 mM p-nitrophenyl-β-D-galactopyrannoside (ONPG),

0.7 % (v/v) Triton X-100, 150 mM citrate buffer (40 mM citric acid, 10 mM sodium

citrate dehydrate, pH3.5)) was added to 10 µl bead phagosome samples distributed in

a 96-well plate. Samples were incubated at 37 °C and the reaction was stopped by

addition of 150 µl 0.5 M sodium carbonate solution after 1 h. This assay was

performed with three technical replicates. Absorbance at 405 nm (A405) was

determined using a microplate absorbance reader. Enzyme activities were normalized

to the iron-concentration.

2.2.1.7.2 Ferrozoin assay

To quantify the amount of magnetic beads in phagosome samples, the iron content

was used. The beads are 2.8 µm uniform, superparamagnetic, polystyrene beads

coated with a polyurethane layer and since they harbor definite amounts of iron, the

iron-concentration in the sample can be directly linked to the number of bead

phagosomes.

10 µl purified control and TDM bead phagosomes were incubated with 50 µl 12 % HCl

for 4 h at 60 °C. Afterwards, samples were spun down at 12.000 xg for 10 min

Supernatants were collected and distributed in a 96-well plate. Samples were

supplemented with 50 µl 1 % ammonium persulfate (APS) and incubated for 5 min at

RT and 100 µl 0.1 M potassium thiocyanate (KSCN) for 5 min at RT in tandem. Finally

absorbance was measured at 490 nm (A490) using a microplate absorbance reader. In

order to calculate iron concentrations, the assay was performed with 50 µl of two

standard dilution series ranging from 1000 µM to 18.75 µM and 300 µM to 4.6 µM,

respectively and treated likewise.


42

Iron-concentrations of control and TDM bead phagosome samples were calculated

with the help of the standard dilution series using Microsoft Excel 2010. The assay

was performed with three technical replicates per sample.

2.2.2 Methods for proteomic analysis

2.2.2.1 Modifications to chapter 2.2.1 for proteomic analysis

“Bead infection” of RAW264.7 macrophages with control and TDM beads was carried

out pair-wise one pair per day. For proteomic analysis, 4 T75-flasks with 107

RAW264.7 macrophages were prepared for one sort of bead (control and TDM) giving

rise to 8 T75-flasks with 107 cells each to be prepared one day prior the bead infection

experiment. RAW264.7 macrophages were infected with a MOI of 10. Accordingly

4x108 beads were coated with BSA (control) only and 4x108 with TDM as outlined

specifically in 2.2.1.3.

Further processing samples was carried out at the laboratory of our collaboration

partners Prof. Dr. Dörte Becher and Dr. Andreas Otto at the University of Greifswald,

Department of Microbial Proteomics by Jürgen Bartel. Here four pairs of control and

TDM bead phagosomes and their corresponding StrataClean bead precipitated

supernatants (in total 16 samples) were further processed for mass spectrometry

analysis.

2.2.2.2 1D-PAGE

First, samples were dried in a vacuum centrifuge and solved with 20 µl Roti-load. Then

samples were incubated for 5 min at 95 °C, cooled, spun down by centrifugation, and

loaded onto a 4–20 % Criterion-TGX-Precast gel. Electrophoresis was carried out at

150 V. The gel was fixed with 10 % (v/v) acetic acid in 40 % (v/v) ethanol for 15 min

and stained with colloidal Coomassie overnight.

2.2.2.3 Gel-based liquid chromatography coupled to mass spectrometry (GeLC-

MS)

After staining, the gel was washed twice with water to remove excessive Coomassie

stain. The stacking gel was removed and all gel lanes of interest were excised. The

lanes were cut into 10 equidistant pieces. Each gel piece was further chopped up into

small cubes of approximately 1 mm3 and transferred into a low binding tube. The gel

pieces were washed/destained at least three times for 15 min with 700 μl of gel wash

buffer (0.2 M ammonium bicarbonate in 30 % (v/v) acetonitrile) at 37 °C under

vigorous shaking. The destained gel pieces were desiccated in a vacuum centrifuge


43

at 30 °C and rehydrated with trypsin solution (2 μg of modified trypsin in 1 ml of water)

for 15 min. Excessive trypsin solution was removed, and the digest was performed

overnight at 37 °C. The gel pieces were covered with water, and the peptides were

eluted from the gel matrix by immersion of the reaction tube in an ultrasonic bath for

15 min. The supernatant containing the peptides was removed, transferred to a glass

vial and concentrated to a final volume of 10 μl in a vacuum centrifuge. For LC–MS/MS

analyses of 1D gel samples, in-house self-packed columns were prepared and used

with an EASY-nLC II system or an Proxeon1000 system with comparable conditions.

In brief, fused-silica emitter tips with an inner diameter of 100 μm and an outer

diameter of 360 μm were prepared by using a P-2000 laser puller. The resulting emitter

tip was then packed with Aeris C18 reversed-phase material (3.6 μm particles) in a

custom-built pressure bomb to obtain a 20 cm nano-LC column.

The peptides were loaded onto the column by the LC system with 10 μl of buffer A

(0.1 % (v/v) acetic acid) at a constant flow rate of 500 nl/min without trapping. The

peptides were subsequently eluted using a nonlinear 85 min gradient from 1 to 99 %

buffer B (0.1 % (v/v) acetic acid in acetonitrile) (Table 2-10) with a constant flow rate

of 300 nl/min and injected online into the mass spectrometer.

Table 2-10: Elution gradient of peptides with buffer B.

Duration [min] Buffer B [%]

1 5

73 25

10 75

1 99

4 99

1 1

10 1

MS and MS/MS data were acquired with a LTQ Orbitrap Velos. After a survey scan at

a resolution of 60 000 in the Orbitrap with activated lockmass correction, the 20 most

abundant precursor ions were selected for fragmentation. Singly charged ions as well

as ions without detected charge states were not selected for MS/MS analysis.

Collision-induced dissociation (CID) fragmentation was performed for 30 ms with

normalized collision energy of 35, and the fragment ions were recorded in the linear

ion trap.


44

2.2.2.4 Data Analysis

Database searching was performed with Sorcerer-SEQUEST 4. After initial extraction

from the proprietary raw files, spectra were searched with Sequest against a target-

decoy database with a set of common laboratory contaminants based on the fasta

sequences for Mus musculus downloaded from NCBI. The resulting peptide-to-

spectrum matches were compiled with the Scaffold 4 software. Proteins were only

considered as identified when at least two unique peptides matching solid quality

criteria (delta cN > 0.1 and XCorr > 2.5, 3.5 and 3.8 for doubly, triply, or higher charged

peptides) have been assigned.

2.2.3 Methods for evaluation of TDM interaction partners

2.2.3.1 Cell culture

One day prior infection with beads or M. tuberculosis GFP, a 24-well plate was

equipped with sterile coverslips (Ø10 mm). 2x105 RAW264.7 macrophages

resuspended in 100 µl D10-media were seeded onto each coverslip to form a drop

and incubated for 1 h at 37 °C and 7.5 % CO2 to let cells adhere to the glass. Finally,

wells were filled up to a total volume of 500 µl with D10-media and incubated ON at

37 °C and 7.5 % CO2.

2.2.3.2 “Infection” of macrophages with lipid coated beads

RAW264.7 macrophages were infected with control or TDM beads in a MOI of 3. D10-

media was discarded and cells were washed once with 500 µl 37 °C PBS.

Subsequently, D10-media containing control or TDM beads was added and

macrophages were incubated for 5 min at 4 °C and 5 min at 37 °C and 7.5 % CO2.

The supernatant containing non-attached beads was removed and cells were washed

once with 500 µl 37 °C D10-media. 500 µl 37 °C D10-media was added and cells were

further incubated at 37 °C and 7.5 % CO2 and stopped at the time points indicated by

fixing coverslips in 500 µl 4 % paraformaldehyde (PFA) ON at 4 °C.

2.2.3.3 Infection of macrophages with M. tuberculosis

2.2.3.3.1 M. tuberculosis GFP culture

Infection experiments with M. tuberculosis GFP were carried out in the biosafety-level

3 laboratory. M. tuberculosis GFP was thawed one week prior infection experiment

and cultivated in 10 ml 7H9-media supplemented with 10 % (v/v) OADC and 5 µg/ml

hygromycin at 37 °C. After four days, the culture was split 1:10 in 10 ml 7H9-media

supplemented with 10 % (v/v) OADC and 5 µg/ml hygromycin and incubated for three

days at 37 °C.


45

2.2.3.3.2 Determination of bacterial number

For infection of macrophages, the M. tuberculosis GFP culture was harvested by

centrifugation at 4000 rpm for 10 min. The supernatant was removed, the pellet was

resuspended in 10 ml PBS and centrifuged at 4000 rpm for 10 min. Again, the

supernatant was removed and the pellet was resuspended in 1 ml PBS. For

separation, the bacterial suspension was pushed through a needle five times. The

number of bacteria was determined by optical density (OD580). Therefore, samples

were diluted 1:20 in 4 % PFA. An OD580 of 0.1 correlated with 5*107 bacteria per ml.

2.2.3.3.3 Macrophage infection

RAW264.7 macrophages were infected with M. tuberculosis GFP in a MOI of 3. D10-

medium was removed and cells were washed once with 500 µl warm PBS.

Subsequently, 200 µl D10-media containing M. tuberculosis GFP was added and

macrophages were centrifuged 5 min at 1500 rpm. Infection was stopped by putting

the coverslips into a new 24-well plate equipped with 500 µl 4 % PFA and incubation

ON at 4 °C.

2.2.3.4 Lysosomal labeling

For staining of lysosomes, 20 min prior stopping the experiment, cells were incubated

with 70 nM lysotracker red.

2.2.3.5 Immunofluorescence staining

Coverslips were placed on parafilm and washed twice with 100 µl PBS. To lower

background fluorescence, coverslips were incubated for 20 min with 70 µl 50 mM

ammonium chloride, washed again twice with 100 µl PBS and blocked and

permeabilized for 15 min with 70 µl 10 % (v/v) goat serum/0.1 % (v/v) Triton X in PBS.

Afterwards, samples were washed twice with 100 µl PBS. Primary antibodies were

incubated 1 h at RT. Samples were washed twice with 100 µl PBS and the secondary

species specific antibody was added for 30 min at RT. Coverslips were washed twice

with 100 µl PBS and stained for DNA with 100 µl 0.5 mg/ml 4',6-diamidino-2-

phenylindole (DAPI) for 10 min at RT. Finally samples were washed twice with 100 µl

PBS and embedded onto microscopic glass slides using confocal matrix. Table 2-11

lists the antibodies used herein and their corresponding dilutions used for

immunofluorescence staining.


46

Table 2-11: Dilutions of primary and secondary antibodies and other labels

Antibodies Final immunofluorescence dilution

Anti-annexinA1 1/1000

Anti-annexin6 1/1000

Anti-cofilin1 1/300

Anti-profilin1 1/300

Anti-SNAP23 1/100

Anti-VAMP3 5 µg/ml

Anti-LAMP1 1/1000

Anti- WASH1 1/150

Anti-rabbit cy2 1/100

Anti-rabbit cy3 1/100

Anti-rat cy5 1/100

Anti-rabbit alexa405 1/100

Dyes Final immunofluorescence dilution

Phalloidin Alexa 488 1/40

Lysotracker red 70 nM

2.2.3.6 Gene knock down by small interference RNA

Cell numbers were determined as described in 2.2.1.2. 2x106 cells were added to a

1.5 ml tube and spun down 10 min at 90 xg at RT. The pellet was resuspended in

100 µl RT Nucleofector-solution. Subsequently, 60 pmol siRNA was added and the

cells were transferred into the transfection cuvette. Transfection was carried out in the

Nucleofector device using program D32. Immediately, 500 µl warm D10-media was

added and cells were transferred to 6-well (control) or 24-well (for experiment) plates

equipped with 1 ml pre-warmed D10-media and incubated for desired time frames at

37°C and 7.5 % CO2 until use. In parallel to siRNAs targeted to desired proteins,

transfection with non-targeting RNA was carried out likewise. For control of siRNA

mediated knock-down of desired proteins, cells were transfected with non-targeting

siRNA, harvested as described above and destroyed by three freeze-thaw cycles

(N2(l), 37 °C H2O). Quantification of protein levels was determined by Western blot.

2.2.3.7 Western blot

2.2.3.7.1 Determination of protein concentration

Protein concentration was determined using the Pierce 660 nm assay: 10 µl of

samples were incubated with 150 µl Pierce reagent for 5 min at RT and the


47

absorbance was determined at 660 nm using a microplate reader. A BSA-based

standard dilution series (2 mg/ml to 0.03125 mg/ml) was treated likewise. Protein

concentrations were calculated by the software of the microplate reader.

2.2.3.7.2 SDS-PAGE

For SDS-PAGE, 10 µg protein of control and TDM bead phagosomes were mixed with

Roti-load, incubated 5 min at 95 °C, spun down shortly and loaded onto a 10 % SDS-

gel. The gel was run for 1-1.5 h at 100 V with electrode running buffer (3.03 g tris base,

14.4 g glycine, 1 g SDS for 1 l) using the Biorad Mini-PROTEAN Tetra Cell system.

Table 2-12: SDS-PAGE gel formulations.

Ingredient Volume [ml] for stacking gel (4 %)

Volume [ml] for resolving gel (10 %)

Deionized water 6.1 4.1

30 % Acrylamide/Bis Solution, 19:1 1.3 3.3

1.5 M TRIS/HCl, pH8.8 - 2.5

0.5 M TRIC/HCl, pH6.8 2.5 -

10 % SDS 0.1 0.1

TEMED 0.01 0.005

10 % APS 0.05 0.05

2.2.3.7.3 Blotting

For Western blotting, gels were placed onto a PVDF-membrane and blotted for 45 min

at 100 V in transfer buffer (25 mM tris, 192 mM glycine, 20 % (v/v) methanol, pH8.3)

using the Biorad Mini Trans-Blot Cell system. The PVDF-membranes were blocked to

prevent unspecific antibody binding by incubation with blocking buffer (5 % (v/v) milk

powder, 0.05 % (v/v) Tween 20 in PBS) ON at 4 °C with gentle shaking. On the next

day, membranes were incubated with the primary antibodies in 8 ml blocking buffer

according to Table 2-13 ON at 4 °C with gentle shaking.

Table 2-13: Concentrations of antibodies used for Western blot.

Antibodies Final dilution for Western blot

Anti-annexinA1 1/1000

Anti-annexin6 1/1000


48

Anti-cofilin1 1/5000

Anti-profilin1 1/20000

Anti-SNAP23 1/2500

Anti-VAMP3 1 µg/ml

Anti-α-tubulin 1/1000

anti-rabbit HRP 1/5000

Membranes were washed thrice with 10 ml blocking buffer for 10 min under gentle

shaking. The secondary antibody coupled to horseradish peroxidase (HRP) was

applied for 3 h at RT in 8 ml blocking buffer. Finally, membranes were washed thrice

with 10 ml blocking buffer for 10 min under gentle shaking and rinsed shortly with

deionised water. Subsequently, membranes were incubated with 2 ml HRP ECL

chemiluminescent detection reagent for 1 min at RT. Finally, blots were placed into

autoradiography cassettes, exposed for different time points (15 sec to 2 min) and

developed. Developed films were scanned with a densitometer to obtain high

resolution digital pictures.

2.2.3.8 Colony forming unit assay

M. tuberculosis GFP was grown as described in 2.2.3.3. For CFU analysis, 2x105

RAW264.7 macrophages per well were infected with M. tuberculosis GFP in a MOI of

0.5. D10-media was removed and cells were washed once with 500 µl PBS.

Subsequently, 200 µl D10-media containing M. tuberculosis GFP was added and

macrophages were centrifuged 5 min at 1500 rpm and incubated at 37 °C and 7.5 %

CO2. After 2 h post infection, the media was removed, cells were washed with 200 µl

D10-media and incubated with 500 µl fresh D10-media at 37 °C and 7.5 % CO2. CFU

were analyzed at desired time points after infection. Therefore, the D10-media was

removed, cells were washed with 200 µl PBS and disrupted by addition of 100 µl 0.5 %

(v/v) Triton X 100 in PBS. Subsequently, a dilution series from 10-1 to 10-5 was

prepared in 0.05 % (v/v) Tween 80 in PBS. 50 µl of dilutions 10-2 to 10-5 were streaked

out onto 7H11-agar/10 % (v/v) cattle serum plates and incubated at 37 °C for 3-4

weeks.

2.2.4 Methods for lipidomic analysis

2.2.4.1 Modifications to chapter 2.2.1 for lipidomic analysis

Control and TDM bead infection of RAW264.7 macrophages was carried out pair wise

one pair per day. For lipid analysis 2 T75-flasks with 107 RAW264.7 macrophages

were prepared one day prior the bead infection experiment for one sort of bead giving


49

rise to 4 T75-flasks with 107 cells each. RAW264.7 macrophages were infected with a

MOI of 10. Accordingly 2x 108 control and TDM beads were prepared as outlined

specifically in 2.2.1.3. Infection of RAW264.7 macrophages with control and TDM

beads and subsequent isolation and purification of bead phagosomes was carried out

as described in 2.2.1, except that D10-media lacking FCS was used.

The data shown here derive from two separate efforts to determine the lipid

composition of bead phagosomes. In the first experiment, sample volumes of three

control and TDM bead phagosome pairs corresponding to 4.000 pM iron were

extracted. In the second attempt, sample volumes of four pairs corresponding to

15.000 pM iron were used. Increasing the amount of source material was necessary

to increase signal intensities of cholesterol in acetylated extracts as they were found

to be very low in the first experiment.

Analysis of lipids was performed in the “Bioanalytical Chemistry” laboratory of Dr.

Dominik Schwudke together with Dr. Nicole Zehethofer.

2.2.4.2 Lipid extraction from isolated and purified lipid coated bead

phagosomes

All glass pipettes and glass vials used for lipid extraction were washed three times with

water, methanol and methyl tert-buthyl ether (MTBE) prior to use. First sample

volumes corresponding to 4.000 pM and 15.000 pM iron were transferred to 2 ml

Eppendorf tubes and dried 4-6 h at 37 °C and 500 rpm in a vacuum centrifuge.

Subsequently, 50 µl water, 270 µl methanol and 50 µl internal standard mix (Table

2-14) were added and mixed thoroughly.

Table 2-14: Internal standard mix

Lipid Concentration [fmol/µl]

17:0-14:1 PC 1.346

17:0-20:4 PC 1.090

17:0-14:1 PE 1.613

17:0-14:1 PG 1.546

17:0-14:1 PS 1.360

17:0-14:1 PI 1.272

Sphingomyelin (d18:1/12:0) 1.230

C25 Ceramide 1.320

16:0-17:0 BMP (R,R) 1.332


50

17:0 Lyso PC 1.420

Cholesterol D7 1.270

Then 1 ml MTBE was added and samples were incubated shaking at 600 rpm for 1 h

at RT. For phase separation, 250 µl water was added and samples were spun for 1 min

at 12.000 rpm. The upper organic phase (circa 800 µl) was transferred to a new 2 ml

Eppendorf tube and dried 1-1.5 h at 37 °C in a vacuum centrifuge. Samples were

dissolved in 100 µl chloroform/methanol (86/13, v/v). 50 µl were transferred to an LC

vial for analysis. 50 µl were saved for cholesterol measurements. Negative controls

comprised uncoated, BSA-coated, TDM-coated and cell free assay BSA-coated and

TDM-coated beads and were treated likewise.

2.2.4.3 Derivatization of cholesterol

Cholesterol measurements were performed as described in [99]. Briefly, due to its

neutral chemical nature, cholesterol had to be acetylated in order to be ionized for

mass spectrometry analysis. All glass pipettes and glass vials used for acetylation

were washed three times with water, methanol and chloroform prior to use. First,

samples were transferred to a glass vial and dried in a vacuum centrifuge 1-1.5 h at

37 °C. Derivatization was performed by addition of 200 µl acetylchloride/chloroform

(1/5, v/v) and incubation for 1 h at RT under a fume hood in an open glass vial.

Afterwards, residual acetylation reagent was removed by centrifugation in a vacuum

centrifuge 1-1.5 h at 37 °C. Until use, samples were stored at -20 °C.

2.2.4.4 µLC-FT-ICR-MS

An Agilent 1100 HPLC system was used for the separation of the phospholipids on a

150 mm BETASIL Diol-100 column with a particle size of 5 µm and 0.32 mm inner

diameter using 5 µl injection volumes. The solvents, gradient profile and flow rates are

shown in Table 2-15.

Table 2-15: Gradient and flow rates used for the separation of phospholipids by µLC-FT-ICR-MS

Time [min] Flow rate [µl/min] Solvent A [%] Solvent B [%]

0 5 0 100

5 5 0 100


51

25 5 58 42

29 5 58 42

30 5 58 42

35 10 58 42

36 10 70 30

40 10 70 30

41 10 0 100

55 10 0 100

Solvents: A, methanol/ammonia solution 28-30 % (99/1, v/v), B, chloroform/methanol/ammonia solution 28-30 % (86/13/1, v/v/v)

All mass spectrometric analyses were performed on a high resolution Bruker Apex Qe

FT-MS equipped with a 7 Tesla actively shielded magnet and an Apollo Dual ESI/

MALDI ion source. For lipid analysis, full scan MS data were acquired in the positive

and negative ion mode in the first 50 min of the liquid chromatography (LC) separation.

Instrumental parameters were as shown in Table 2-16. Data acquisition was

performed using the HyStar 3.2 software.

Table 2-16: FT-ICR-MS instrumental settings

Parameter Positive ion mode Negative ion mode

data points 1 M 1 M

accumulation time 0.9 seconds 1.1 seconds

average spectra 1 1

source temperature 200 °C 200 °C

ionization voltage 4500 kV 3800 kV

Q1 mass 300 m/z 250 m/z

collision energy 0 V 0 V

TOF time 0.0014 0.0014

nebulising gas 1 l/min 1 l/min

dry gas 5 l/min 3 l/min

2.2.4.5 ESI Qq-TOF-MS

Cholesteryl acetate was measured using an ESI Qq-TOF-MS operating in the parallel

reaction monitoring (PRM) mode. Prior to analysis, dried samples were dissolved in

100 µl MS-mix (1/2/4, v/v/v, chloroform, methanol containing 0.1 % ammonium

acetatel, 2-propanol). For flow injection analysis, 20 µl of the mixture were injected into

the ESI Qq-TOF-MS via an Agilent 1100 HPLC system. The solvent used and flow

rates are shown in Table 2-17.


52

Table 2-17: Flow rates used for the analysis of cholesteryl acetate by ESI Qq-TOF-MS

Time [min] Flow [µl/min] Pressure [bar]

0 5 400

8 5 400

8.01 20 400

Solvent: MS-mix

Source temperature was set to 150 °C and instrumental parameters were optimized

prior analysis following recommendations of the manufacturer. PRM scans of m/z 446

and m/z 443 were performed using 1 Da isolations width and CID voltages of 10 V and

10.3 V for Chol-D7 and Chol, respectively.

2.2.4.6 Data processing and analysis

µLC-FT-ICR-MS data files were processed using Data Analysis 4.0 software. Mass

spectra were averaged in the retention time ranges of lipid classes of interest (defined

by the elution time of the internal standards). Each mass spectrum was smoothed,

baseline subtracted and peaks lists were extracted (m/z values and corresponding

intensities). Lipids were identified by their monoisotopic masses with an accuracy

better 16 ppm (LBPA) and 3 ppm (all others) using LipidXplorer [97] and their

respective retention time window.

Intensity ratios of lipid species and their corresponding internal standard were used for

quantification of lipids in the sample. All further calculations were performed with

Microsoft Excel 2010. First samples were grouped to BSA (control), TDM and negative

controls. Lipids with intensities higher than 10 % in negative controls compared to BSA

and TDM samples were deleted. For minimal occupation requirements, lipids had to

be present in at least half of the BSA or half of the TDM samples. To calculate the

amount of lipids in pmol, intensities of identified lipids were divided by the intensity of

the corresponding internal standard and multiplied by the amount of the internal

standard added.

For further analysis, values for all identified species obtained from both experiments

were combined. To standardize all samples to the amount of bead phagosomes used

in the experiment, pmol values were divided by the amount of iron in µM in the

corresponding sample. Then, the amount of all lipids in one sample was summed up.

For calculation of the mol percentages (mol %), the amount of every lipid species in


53

pmol was divided by the sum of all lipids identified in the samples and multiplied by

100.

ESI Qq-TOF-MS data were processed using the MassLynx 4.0 software. Intensity

values were obtained by manually combining mass spectra observed above half peak

height (spectra #3-59) in PRM scans of m/z 446 and m/z 443, respectively. Intensities

of peaks corresponding to the product ion masses of cholesteryl acetate (m/z 369) and

the deuterated standard (m/z 376) were picked and used for calculations. To calculate

the amount of free cholesterol, the intensity ratio of cholesteryl acetate and the

corresponding deuterated internal standard was used.

2.2.4.7 Statistical analysis and graphic representation

For statistical analysis and graphic representation, the Graph Pad Prism 5 software

was used. For statistical analysis of lipid classes, the mol % of every lipid class was

summarized. Values for every sample pair were analyzed with a paired TTest and

depicted with a before-and-after plot.

For statistical analysis of lipid species, as well, mol % values were used. For every

lipid class and corresponding samples pair, p-values were calculated using a paired

TTest. Further, the ratios between every sample pair were calculated by dividing the

mol % of TDM samples by the mol % of control samples. The mean of all seven p-

values for every lipid species was transformed to a log10-value and multiplied by -1.

The mean of all seven ratios for every lipid species was transformed to a log2-value.

For volcano-plot analysis, the transformed ratios were plotted against the transformed

p-values.

Results

54

3 Results

3.1 Method development: Isolation and purification of bead

phagosomes for mass spectrometry analysis

Inhibition of phagosome maturation by TDM is well established, but the underlying

mechanisms at the phagosomal interface remain unknown [90], [91], [100].

Consequently an experimental system to study the effect of single lipid species on

phagosome maturation and concomitantly to identify potential host-cell interaction

partners was required. In 2008, Axelrod et al. developed a reductionist lipid-coated

bead model [91]. Lipids such as TDM were coated to beads and fed to macrophages

to monitor bead phagosome maturation using stage specific markers.

To achieve the prime aim of this study that is identification of host-cell derived putative

interaction partners, bead phagosome proteomes and lipidomes had to be analyzed

by mass spectrometry. Based on the model system described by Axelrod et al., an

optimized protocol to isolate and purify bead phagosomes from macrophages was

developed.

To identify TDM specific interaction partners, control and TDM bead phagosomes were

isolated and purified for comparative proteomic and lipidomic analysis. Thus, magnetic

beads were coated with BSA (control) and TDM and used for “bead infection” of

macrophages. After 30 min, bead phagosome maturation was stopped by scraping the

cells in ice-cold buffer and disruption using a metal douncer. The disruption process

released un-cleaved DNA which caused formation of aggregated vesicles. Therefore,

as a first purification step, samples were treated with DNase to eliminate DNA. Further,

samples were washed three times using a magnet. Since the bead phagosomes still

stuck together, samples were additionally treated with trypsin. To separate bead

phagosomes from cell debris, a Ficoll gradient was applied. Differences in phagosome

maturation were controlled by measuring lysososmal β-galactosidase activity in the

samples prior continuing the purification procedure. This step had to be performed

prior sorting, because after sorting process bead phagosome concentrations were too

low to yield a signal in the lysososmal β-galactosidase assay. As final step, bead

phagosomes were sorted by FACS to separate bead phagosomes from residual cell

debris. Bead phagosomes were isolated from the FACS supernatant using a magnet

and proteins remaining in the supernatant were precipitated using StrataClean beads.

A scheme of the overall practical procedure for isolation and purification of bead

phagosomes is shown in Figure 3.1.

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Figure 3.1: Scheme of the procedure for isolation and purification of bead phagosomes from macrophages for mass spectrometry analysis. After coating of magnetic beads with TDM (step 2), macrophages were “bead infected” with control or TDM beads (step 3). Following 30 min of bead phagosome maturation, cells were disrupted using a douncer at 4 °C (step 4). Bead phagosomes were purified by DNase digestion (step 5) and freed from crude cell debris by magnetic isolation (step 6) and further, trypsin (step 7) treatment and Ficoll gradient centrifugation (step 8). Finally bead phagosomes were freed from residual cell debris by sorting by FACS (step 9) and analyzed for their protein content by mass spectrometry. β-galactosidase activity of control and TDM bead phagosomes was determined prior sorting by FACS.

3.1.1 Quality control of purified bead phagosomes

Potential interaction partners were predicted to be found exclusively in or associated

with bead phagosomes that halted a non-mature stage by TDM. Thus TDM mediated

inhibition of bead phagosome maturation had to be tested prior analysis of samples by

mass spectrometry. As mentioned above prior sorting by FACS, β-galactosidase

activity was used to reveal the phagosomal stage. Active β-galactosidase is only found

in late endosomal/lysosomal compartments. Thus, phago-lysosomes contain more

enzymatic activity than early, intermediate or late phagosomes. Consequently, due to

TDM-mediated inhibition of phagosome maturation, TDM bead phagosomes should

have less β-galactosidase activity compared to control ones. The β-galactosidase

activity of all four sample pairs analyzed in this study is shown in Figure 3.2. TDM bead

phagosomes of samples 1 to 4 (A-D), showed less β-galactosidase activity (89.22 %,

87.46 %, 60.85 % and 54.15 %) compared to controls. Although TDM was able to

inhibit maturation of bead phagosomes compared to control ones, the differences

between TDM and control bead phagosomes differed significantly between

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preparations. All four sample pairs were used further purification and mass

spectrometry analysis.

Figure 3.2: β-galactosidase assay of purified control versus TDM bead phagosomes. A-D: Macrophages were “bead infected” with control and TDM beads at a MOI of 10. Bead phagosome maturation was allowed for 30 min. Afterwards, cells were harvested and bead phagosomes were isolated and purified as described in detail in section 2.2.1. Prior to sorting by FACS, 150 µl of control and TDM bead phagosomes in PBS were saved for verification of β-galactosidase activity as described in 2.2.1.7. The β-galactosidase activity was standardized with the number of beads per sample.

3.1.2 Purification of bead phagosomes via sorting by FACS

The efficiency of the final purification step by FACS is shown in Figure 3.3. After

sorting, TDM and control bead phagosome samples were reanalysed for purity and

presence of remaining contaminating cell debris. Representatively, prior sorting, in

control bead isolation number 4 and in TDM bead phagosome isolation number 3,

86.23 % and 93.22 % of all detectable particles were bead phagosomes, respectively.

However, sorting by FACS increased the purity of the samples to 97.72 % and

98.88 %, respectively. Concluding, sorting by FACS was suitable to exclude

contaminants and increase purity of isolated bead phagosomes.

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Figure 3.3: Control and TDM bead phagosomes prior to and after sorting by FACS. Representative fraction of bead phagosomes enriched by a FACSAriaII cell sorter from TDM bead phagosome isolation #3 (lower) and control bead isolation #4 (upper). The Area of Forward (FSC-A) and Side Scatter (SSC-A) was used to determine the bead fraction. Before sorting, the portion of bead was around 93.22 % (TDM) and 86.23 % (control) (left scatter plot), after sorting and re-analyzing the sorted fraction, the enrichment was increased up to 98.22 % and 97.72 % purity of bead phagosomes (right scatter plot), respectively.

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3.2 The TDM bead phagosome proteome

Processing of all sample pairs for mass spectrometry analysis was performed in the

laboratory of our collaboration partners Prof. Dr. Dörte Becher and Dr. Andreas Otto

in Greifswald by Jürgen Bartel as described in detail in 2.2.2. The data obtained that

way, were received as “Scaffold file” and further processed with the “Scaffold 4”

software.

In total 1054 proteins were detected in all four sample pairs (Table 0-1). In control and

TDM bead phagosome samples, 919 and 843 proteins were detected, respectively.

As shown in Figure 3.4, 708 proteins were shared between both groups while 211 and

135 proteins were detected exclusively either in control or TDM bead phagosome

samples, respectively.

Figure 3.4: Intersection diagram of total numbers of identified proteins in proteomes of control versus TDM bead phagosome samples. Four pairs of isolated and purified control and TDM bead phagosomes samples were analyzed for their protein content by mass spectrometry. Results were processed using the “Scaffold 4” software. 1054 proteins were identified in total. 708 proteins were shared between control and TDM bead phagosome proteomes whereas 211 proteins were found exclusively in control and 135 in TDM bead phagosomes, respectively.

Using the Scaffold 4 software, the NCBI annotations were downloaded that provide

current information about each identified protein. Figure 3.5 depicts all proteins

grouped according to their cellular localization according to the NCBI definition.

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Figure 3.5: Identified proteins in control and TDM beads phagosomes grouped according to their cellular localization. Four pairs of isolated and purified control and TDM bead phagosomes samples were analyzed for their protein composition by mass spectrometry. Results were processed using the “Scaffold 4” software. Using the “quantify” mode, all identified proteins were sorted according to their cellular localization. The numbers shown above for each category exceed the total numbers of 1054 identified proteins because some proteins were allocated to several cellular localizations.

Accordingly, 684 and 512 proteins were associated with intracellular organelles and

the organelle part (same as intracellular organelle, term should not be used any more

according NCBI but has not been replaced at the time this thesis was prepared),

respectively. 632 were cytoplasmic proteins. Of the 478 membrane proteins, 294 and

229 were allocated to organelle or plasma membranes, respectively. 288 proteins

were grouped to the nucleus. 173 mitochondrial proteins were discovered. Further, of

the endoplasmic reticulum and the cytoskeleton 123 proteins were enriched whereas

87 proteins were assigned to the Golgi apparatus and the endosome. 21 ribosomal

and 83 extracellular proteins were identified. Finally, 264 proteins had unknown

localization.

Proteins specified with the categories ribosome, extracellular region, Golgi apparatus,

cytoskeleton, ER, mitochondria, nucleus and cytoplasm are potential contaminant

proteins, because per definition of the gene ontology (GO) terms, these groups do not

comprise proteins that localize in intracellular vesicles or their membranes. In contrast,

categories endosome, intracellular organelle, organelle part or membrane and

organelle membrane can contain, per GO term definition, proteins within organized

structure of distinctive morphology and function, occurring within the cell or associated

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with membranes thereof. These categories may be those containing phagosomal or

lysosomal proteins.

Next, the percentage of proteins according to their cellular localization in control versus

TDM bead phagosome was compared. Figure 3.6 shows that there were no major

differences between both samples.

Figure 3.6: Percentage distribution of all proteins identified in control versus TDM bead phagosome samples grouped according to their cellular localization. Four pairs of isolated and purified control and TDM bead phagosome samples were analyzed for their protein content by mass spectrometry. Results were processed using the “Scaffold 4” software. The “quantify” mode of the software sorts all identified proteins according to their cellular localization and calculates the percentages of identified proteins that can be classified to a cellular compartment. The numbers shown above for every category exceed 100 because some proteins have more than one cellular localization.

Identified proteins were quantified by spectral counting. Using the Scaffold 4 software,

the exclusive unique spectrum count (EUSC) mode was selected to assess the

“number of unique spectra associated only with this protein”. For further calculations,

the EUSC of all proteins identified in all four sample pairs was exported from Scaffold

4 to a Microsoft Excel file. In order to find proteins that were more abundant in TDM

bead phagosome samples compared to controls, and thus are potential TDM host-cell

derived interaction partners, first all proteins with an overall EUSC less than 10 were

excluded. For minimal occupation requirements, proteins had to be identified in at least

4 of the 8 control or TDM bead phagosome samples. Subsequently the EUSC for all

proteins identified in the 8 control and TDM bead phagosomes samples were summed

up and the value of all EUSC from TDM phagosomes was divided by the number of

all EUSC from control phagosomes. Proteins with ratios of ≥2 were considered

enriched to TDM bead samples. Thus, the list of TDM specific proteins comprised 34

(Table 3-1). Proteins with ratios of ≤0.5 were considered enriched in control bead

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62

Synaptic vesicle membrane protein VAT-1 homolog

sp|Q62465|VAT1 6 12 2.0

ATP-dependent RNA helicase DDX18

sp|Q8K363|DDX18 6 12 2.0

Unconventional myosin-Ie sp|E9Q634|MYO1E 6 12 2.0

V-type proton ATPase subunit F

sp|Q9D1K2|VATF 4 8 2.0

Table 3-2: List of proteins enriched in control bead phagosomes. 1054 proteins were identified in control and TDM bead phagosomes. To unravel potential host-cell derived TDM interaction partners, all proteins with an overall EUSC less than 10 were excluded. Next, for minimal occupation requirements, proteins had to be identified in at least 4 of 8 control or TDM bead phagosomes samples. Finally the EUSCs of all control and all TDM bead phagosome samples were added and TDM/control ratios were calculated. Proteins with ratios ≤0.5 were considered enriched in control bead samples.

Identified protein accession number

EUSC control

EUSC TDM

ratio

Cytochrome b-245 heavy chain sp|Q61093|CY24B 14 7 0.5

ER-resident protein 29 sp|P57759|ERP29 14 7 0.5

MO colony-stimulating factor 1 receptor

sp|P09581|CSF1R 8 4 0.5

Arfip1 protein tr|A2RSX9|A2RSX9

15 7 0.5

Desmoplakin sp|E9Q557|DESP 156 70 0.4

Pre-mRNA-processing factor 6 sp|Q91YR7|PRP6 16 7 0.4

Prelamin-A/C sp|P48678|LMNA 22 9 0.4

Triosephosphate isomerase sp|P17751|TPIS 10 4 0.4

14-3-3 protein epsilon sp|P62259|1433 15 6 0.4

Fructose-bisphosphate aldolase tr|A6ZI44|A6ZI44 18 6 0.3

Protein Sec14l1 tr|A2A9B9|A2A9B9 18 4 0.2

Isoform 3 of Poly(rC)-binding protein 2

sp|Q61990-|PCBP2

14 3 0.2

Table 3-3: Cellular localization and biological process of proteins enriched to TDM bead over control phagosomes.

Identified protein Cellular compartment

Biological process

Rab22B OS early endosome / late endosome / phagocytic vesicle / trans-Golgi network / membrane

small GTPase mediated signal transduction / protein transport / receptor internalization / Rab protein signal transduction / cellular response to insulin stimulus / Golgi to plasma membrane protein transport / regulated secretory pathway / Golgi vesicle transport / phagosome maturation

Valine--tRNA ligase

mitochondrium valyl-tRNA aminoacylation

VAMP7 Golgi apparatus / ER membrane / late endosome

vesicle-mediated transport/ Golgi to plasma membrane

http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0032588

http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0032588

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membrane / phagocytic vesicle / cell junction / lysosomal membrane /

protein transport/ eosinophil and neutrophil degranulation

Rab-11A Golgi apparatus / ER membrane / late endosome membrane / phagocytic vesicle / cell junction / lysosomal membrane

small GTPase mediated signal transduction / cytokinesis / protein transport / neurite development / melanosome transport

Protein 5730469M10Rik

cytoplasm / mitochondrium extracellular exosome

regulation of osteoclast differentiation / oxidation-reduction process

Annexin A1 mitochondrial membrane / cornified envelope / extracellular vesicular exosome / nucleus / basolateral plasma membrane / nucleus /

neutrophil homeostasis / alpha-beta T cell differentiation / gliogenesis / insulin secretion / peptide cross-linking / estrous cycle phase / response to drug

Cofilin-1 extracellular region / nucleus / cytoplasm / cytoskeleton / plasma membrane / cell-cell junction / focal adhesion / actin cytoskeleton / membrane / nuclear matrix / lamellopodium /cortical actin cytoskeleton / cell leading edge / vesicle / ruffle membrane

mitotic cytokinesis / neural crest cell migration / neural fold formation / protein phosphorylation / protein import into nucleus / cytoskeleton organization / actin filament organization / response to virus / regulation of cell morphogenesis / negative regulation of cell size / regulation of dendritic spine morphogenesis / response to amino acid / positive regulation of actin filament depolymerisation /

Profilin-1 cytoplasm / cytoskeleton / extracellular region / nucleus

neural tube closure / sequestering of actin monomers

Annexin A6 cytoplasm / mitochondrium / lysosomal membrane / cytosol / focal adhesion / membrane / late endosome membrane / melanosome / perinuclear region of cytoplasm / extracellular exosome /

calcium ion transport / regulation of muscle contraction / ion transmembrane transport / protein homooligomerization / mitochondrial calcium ion homeostasis / apoptotic signalling pathway

TOM34 mitochondrion / mitochondrial outer membrane /

protein targeting to mitochondrion /

60S ribosomal protein L22

cytoplasm / ribosome / cytosolic large ribosomal subunit / ribosome

alpha-beta T cell differentiation / translation

VAMP 3 secretory granule / recycling endosome / cytoplasmic vesicle / plasma membrane / clathrin coated vesicle membrane

substrate adhesion-dependent cell spreading / positive regulation of receptor recycling / neurotransmitter secretion / substrate

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64

adhesion-dependent cell spreading / calcium ion-dependent exocytosis /

SNAP23 nucleoplasm / cytoplasm / plasma membrane / focal adhesion / membrane / cell junction / SNARE complex / cytoplasmic vesicle / specific granule / mast cell granule / neuron projection / synapse / extracellular exosome

histamine secretion by mast cell / transport / exocytosis / protein transport / synaptic vesicle priming / synaptic vesicle fusion to presynaptic membrane

Cytochrome c oxidase subunit 4 isoform 1, mitochondrial

mitochondrion / mitochondrial inner membrane / nucleus

response to nutrient

Protein ERGIC-53 Golgi apparatus / ER membrane / ER to Golgi transport vesicle / ER-Golgi intermediate compartment membrane / Golgi membrane

positive regulation of organelle organization / ER to Golgi vesicle-mediated transport /

Voltage-dependent anion-selective channel protein 3

mitochondrion / mitochondrial outer membrane / pore complex / mitochondrial inner membrane

nerve-nerve synaptic transmission / behavioural fear response / learning

MKIAA1699 protein (Fragment)

exocyst / cytoplasm / membrane / growth cone membrane

vesicle docking involved in exocytosis / protein transport

Signal recognition particle 68 kDa protein

signal recognition particle, endoplasmic reticulum targeting / nucleolus

SRP-dependent cotranslational protein targeting to membrane / response to drug

Pescadillo homolog

condensed chromosome / nucleus / nucleoplasm / chromosome / nucleolus / cytoplasm / membrane / preribosome

maturation of LSU-rRNA from tricistronic rRNA transcript / SSU-rRNA, 5.8S rRNA, LSU-rRNA / rRNA processing / nucleolus organization / cell proliferation / protein localization to organelle / ribosome biogenesis / ribosomal large subunit biogenesis / regulation of cell cycle

Elongation factor 2 cytoplasm translational elongation / hemopoietic progenitor cell differentiation / GTP catabolic process /

Neutrophil cytosol factor 2

signal recognition particle, endoplasmic reticulum / nucleolus

SRP-dependent cotranslational protein targeting to membrane / response to drug

Cytosolic phospholipase A2

Golgi apparatus / ER / cytoplasmic membrane-bounded vesicle

regulation of cell proliferation / phospholipid catabolic process / arachidonic acid

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65

secretion / icosanoid biosynthetic process

Ras-related protein Rab-8A

Golgi apparatus / recycling endosome membrane / postsynaptic density / recycling endosome membrane / phagocytic vesicle membrane / plasma membrane

small GTPase mediated signal transduction / cellular response to insulin stimulus / axonogenesis / Golgi vesicle fusion to target membrane /

Lysosomal protective protein

nucleoplasm / mitochondrium / lysosome / membrane / intracellular membrane-bounded organelle / extracellular exosome

proteolysis

Ras-related protein Rab-10

Golgi apparatus / ER membrane / recycling endosome / endosome / plasma membrane / phagocytic vesicle membrane / recycling endosome membrane /

small GTPase mediated signal transduction / cellular response to insulin stimulus / polarized epithelial cell differentiation / Golgi to plasma membrane protein transport / axonogenesis /

Nucleolar protein 58

cytoplasm/ nucleolus / Cajal body

snRNP protein import into nucleus /

Histone H4 actin cytoskeleton / nucleoplasm /

negative regulation of megakaryocyte differentiation / nucleosome assembly /

Uncharacterized protein


proteolysis

YLP motif-containing protein 1


proteolysis

Major facilitator superfamily domain-containing protein 1

integral to membrane transmembrane transport

Synaptic vesicle membrane protein VAT-1 homolog

mitochondrial outer membrane

negative regulation of mitochondrial fusion

ATP-dependent RNA helicase DDX18

nucleolus

Unconventional myosin-Ie

cytoplasm / myosin complex / adherens junction

platelet-derived growth factor receptor signalling / ATP catabolic process / glomerular basement membrane development / endocytosis / hemopoiesis / glomerular filtration

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66

V-type proton ATPase subunit F

vacuolar proton-transporting V-type ATPase complex

ATP hydrolysis coupled proton transport

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3.2.1 Introduction of selected proteins and STRING analysis

To see whether and how the 34 enriched proteins interact, they were analyzed with

the STRING 9.1 online tool. STRING (Search Tool for the Retrieval of Interacting

Genes/Proteins) is a database of direct (physical) and indirect (functional) predicted

protein interactions. One protein could not be identified since its entry in the source

(NCBI entries) used by STRING was deleted.

Figure 3.7 shows the interaction network of 33 of the 34 selected proteins. Identified

proteins are illustrated by dots, identified genes by smaller dots with their

corresponding names. Interaction between proteins is illustrated by colour-coded

connecting lines. In general, more lines indicate more profound interaction. The

arrangement of proteins without known interactions in the network is purely random

and does not correspond to their potential function or the genetic organisation.

Figure 3.7: STRING network of proteins enriched in TDM bead phagosomes. 1054 proteins were identified in control and TDM bead phagosomes. To unravel potential host-cell derived TDM interaction partners, all proteins with an overall EUSC less than 10 were excluded. Next, for minimal occupation requirements, proteins had to be identified in at least 4 of 8 control or TDM bead phagosomes samples. Finally the EUSCs of all control and all TDM bead phagosome samples were added and TDM/control ratios were calculated. 34 proteins with ratios equal or greater than 2 were considered enriched in TDM and thus potential interaction partners. 33 proteins were analyzed regarding their interaction using the online tool STRING 9.1.

The STRING network shows 6 (numbers 1-6) major interacting networks. Network 1

comprises 7 proteins and 1 gene which function in membrane organization and protein

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localization to membranes. Network 2 includes 3 proteins that have a role in protein

localization to organelles and are ribonucleoprotein complex binding proteins. Network

3 is made of 3 proteins that take part in cellular respiration and energy metabolism.

Network 4 comprises 2 proteins that interact with the actin cytoskeleton. Network 5

involves 3 proteins that are known to be important for hemopoiesis. Finally, network 6

includes 2 proteins involved in fatty acid biosynthesis.

Of the 34 potential TDM interacting proteins, 6 were chosen for further analysis

regarding localization and function in trafficking and M. tuberculosis infection in

macrophages using fluorescence microscopy, Western blot and siRNA knock-down

experiments, respectively (Table 3-4). Selection of those 6 proteins was based on 3

criteria, (i) known association with endosomes/phagosomes and/or (ii) component of

the 6 major networks (Figure 3.7) and (iii) availability of appropriate antibodies for

analysis with immunofluorescence microscopy and Western blot.

Selected proteins are listed in Table 3-4. AnnexinA1 and A6 are important for

calcium/phospholipid-binding, which promotes membrane fusion during phagosome

maturation. They were also found on mycobacterial phagosomes and were described

to link actin filaments to the phagosome during phagocytosis [101], [102]. Profilin1 and

cofilin1 are known to regulate the actin cytoskeleton dynamics important for

phagosome biogenesis [103]. The synaptosomal-associated protein 23 (Snap23) was

recently shown to regulate phagosome formation and maturation in macrophages

[104]. VAMP3 is s SNARE-protein involved in vesicle transport to and from endosomes

and phagosomes.

Table 3-4: Identified proteins selected for further analysis as potential host-cell derived TDM interaction partners.

Identified proteins Accession Number EUSC control

EUSC TDM

Ratio

Annexin A1 sp|P10107|ANXA1 5 17 3.4

Annexin A6 tr|F8WIT2|F8WIT2 8 25 3.1

Cofilin-1 tr|F8WGL3|F8WGL3 4 13 3.3

Profilin-1 sp|P62962|PROF1 7 22 3.1

Synaptosomal-associated

protein (SNAP23)

tr|Q9D3L3|Q9D3L3 7 17 2.4

Vesicle-associated

membrane protein 3 (VAMP3)

sp|P63024|VAMP3 8 20 2.5

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3.3 Evaluation of TDM interaction partners

First, we analyzed the intracellular localization of the selected proteins with respect to

bead and M. tuberculosis phagosomes in macrophages. Therefore, RAW264.7

macrophages were incubated with control beads, TDM beads or M. tuberculosis GFP.

Bead phagosome maturation was stopped after 30 min and M. tuberculosis GFP after

2 h. Subsequently, infected macrophages were stained using antibodies against

annexinA1, annexinA6, cofilin1, profilin1, SNAP23 and VAMP3. To determine the

maturation status of bead and M. tuberculosis GFP phagosomes, macrophages were

co-stained for LAMP1. LAMP1 is an integral membrane protein highly abundant in

matured phagosomes, late endosomes and lysosomes [36]. Thus it is commonly used

as a marker for late phago-/endosomes and phago-lysosomes. Infected cells stained

for cofilin1 and profilin1 were additionally stained for actin in order to study co-

localization of actin with the nucleation-promoting-factors (NPFs).

Notably, not all antibodies available could be used for immunofluorescence staining of

RAW264.7 macrophages infected with M. tuberculosis GFP because they cross-

reacted with M. tuberculosis GFP (results not shown). According to information

obtained from the manufacturers, these polyclonal antibodies were generated in

rabbits using adjuvants containing mycobacterial components. Consequently, the

antibody to annexinA1 was not used because it stained free M. tuberculosis GFP.

Specific binding of antibodies was tested by incubating RAW264.7 macrophages and

M. tuberculosis GFP with the secondary antibody alone. This revealed that neither

Alexa405 anti-rabbit, Cy2 anti-rabbit, Cy3 anti-rat nor Cy5 anti-rabbit displayed

unspecific binding to RAW264.7 macrophages or M. tuberculosis GFP.

In all experiments, TDM bead phagosomes showed significantly decreased LAMP1

association compared to controls. Thus, TDM successfully inhibited maturation of

bead phagosomes.

3.3.1 Evaluation of the presence of candidate proteins on control bead, TDM

bead and M. tuberculosis phagosomes by immunofluorescence staining

AnnexinA1: Figure 3.8 shows representative immunofluorescence pictures of control

(A-D) and TDM (E-H) bead phagosomes in RAW264.7 macrophages stained for

annexinA1 (green) and LAMP1 (red). Quantitative analysis showed that the annexinA1

staining pattern for both bead types was similar, neither TDM nor control bead

phagosomes accumulated annexinA1 as assessed by immunofluorescence staining

(Figure 3.8, I).

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Figure 3.8: Immunofluorescence staining of control and TDM bead infected RAW264.7 macrophages for annexinA1 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3. 30 mpi, phagosome maturation was stopped and macrophages were stained using primary antibodies to annexinA1 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A & E: Staining pattern of annexinA1 in macrophages with control and TDM bead phagosomes. B & F: Staining pattern of LAMP1 in macrophages with control and TDM bead phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D & H: Phase contrast. I: Quantitative analysis of annexinA1 and LAMP1 association with control and TDM bead phagosomes. TTest, n=3 biological replicates, p-value * >0.05, ** >0.01, *** >0.001, bar=5 µm

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AnnexinA6: Figure 3.9 shows representative immunofluorescence pictures of control

(A-D), TDM (E-H) bead and M. tuberculosis GFP (I-L) phagosomes in RAW264.7

macrophages stained for annexinA6 (green) and LAMP1 (red). Quantitative analysis

revealed that neither bead nor M. tuberculosis GFP phagosomes accumulated

annexinA6 as assessed by immunofluorescence staining (Figure 3.9, M)

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Figure 3.9: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for annexinA6 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis GFP with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to annexinA6 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of annexinA6 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of annexinA6 and LAMP1 association with control, TDM bead and M. tuberculosis GFP phagosomes. TTest, n=3 biological replicates, p-value * >0.05, **

>0.01, *** >0.001, bar=5 µm

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Cofilin1: Figure 3.10 shows representative immunofluorescence pictures of control

bead (A-D), TDM (E-H) bead and M. tuberculosis (I-L) phagosomes in RAW264.7

macrophages stained for cofilin1 (green) and LAMP1 (red). Quantitative analysis

showed that the cofilin1 staining pattern for both bead types was similar since

punctuate pattern were absent from control and TDM bead phagosomes. In contrast,

phagosomes of M. tuberculosis GFP show a significant punctuate association with

cofilin1 when LAMP1-positive (arrow). Further, TDM bead and M. tuberculosis

phagosomes were significantly associated with actin compared to controls (not

shown). However, actin did not co-localize with cofilin1. In conclusion,

immunofluorescence staining revealed association of cofilin1 only with LAMP1-

positive M. tuberculosis but not with bead phagosomes or actin.

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Figure 3.10: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for cofilin1 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis GFP with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to cofilin1 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of cofilin1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of cofilin1 and LAMP1 association with control, TDM bead and M. tuberculosis

GFP phagosomes. TTest, n=3 biological replicates, p-value * >0.05, ** >0.01, *** >0.001, bar=5 µm

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Profilin1: Figure 3.11 shows representative immunofluorescence pictures of control (A-

D), TDM (E-H) bead and M. tuberculosis GFP (I-L) phagosomes in RAW264.7

macrophages stained for profilin1 (green) and LAMP1 (red). The profilin1 staining

pattern for both bead types was similar, as the candidate protein was completely

absent from control and TDM bead phagosomes as analyzed by immunofluorescence

staining. However, as with cofilin1, phagosomes of M. tuberculosis GFP show a

significant association with profilin1 when LAMP1-positive (arrow). Further, TDM bead

and M. tuberculosis phagosomes were significantly associated with actin compared to

controls (not shown). However, actin did not co-localize with profilin1. In conclusion,

immunofluorescence staining revealed association of profilin1 only with LAMP1-

positive M. tuberculosis but not with bead phagosomes or actin.

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Figure 3.11: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for profilin1 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis GFP with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to profilin1 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of profilin1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of profilin1 and LAMP1 association with control, TDM bead and M. tuberculosis GFP phagosomes. TTest, n=3 biological replicates, p-value * >0.05, ** >0.01, ***

>0.001, bar=5 µm

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SNAP23: Figure 3.12 shows representative immunofluorescence pictures of control

(A-D), TDM (E-H) bead and M. tuberculosis GFP (I-L) phagosomes in RAW264.7

macrophages stained for SNAP23 (green) and LAMP1 (red). Quantitative analysis

revealed that neither bead nor M. tuberculosis GFP phagosomes accumulated

SNAP23 (Figure 3.12, M). In conclusion, as assessed by immunofluorescence

staining, SNAP23 did not reveal a significant association with bead or M. tuberculosis

GFP phagosomes.

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Figure 3.12: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for SNAP23 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to SNAP23 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of SNAP23 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of SNAP23 and LAMP1 association with control, TDM bead and M. tuberculosis phagosomes. TTest, n=3 biological replicates, p-value * >0.05, ** >0.01, *** >0.001, bar=5 µm

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VAMP3: Figure 3.13 shows representative immunofluorescence pictures of control (A-

D), TDM (E-H) bead and M. tuberculosis GFP (I-L) phagosomes in RAW264.7

macrophages stained for VAMP3 (green) and LAMP1 (red). Quantitative analysis

revealed that neither bead nor M. tuberculosis phagosomes were significantly

associated with candidate protein VAMP3 as assessed by immunofluorescence

staining.

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Figure 3.13: Immunofluorescence staining of control, TDM bead and M. tuberculosis GFP infected RAW264.7 macrophages for VAMP3 and LAMP1. Macrophages were “bead infected” with control and TDM beads (*) in a MOI of 3 or M. tuberculosis with a MOI of 5. 30 mpi and 2 hpi, respectively, phagosome maturation was stopped and macrophages were stained using primary antibodies to VAMP3 and LAMP1 followed by secondary fluorescent antibodies to either rabbit or rat IgG, respectively. A, E & J: Staining pattern of VAMP3 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. B, F & K: Staining pattern of LAMP1 in macrophages with control, TDM bead and M. tuberculosis GFP phagosomes. C & G: Merged images with DAPI stain for nucleus (blue). D, H & L: Phase contrast. I: M. tuberculosis GFP. M: Quantitative analysis of VAMP3 and LAMP1 association with control, TDM bead and M. tuberculosis phagosomes. TTest,

n=3 biological replicates, p-value * >0.05, ** >0.01, *** >0.001. bar=5 µm

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3.3.2 Evaluation of the presence of candidate proteins on isolated and purified

control and TDM bead phagosomes by Western blot

To further validate the differential abundances of annexinA1, annexinA6, cofilin1,

profilin1, SNAP23 and VAMP3 found by proteomics, control and TDM bead

phagosomes were isolated and purified from RAW264.7 macrophages and analyzed

by Western blot. For this purpose, the isolation and purification protocol did not include

sorting by FACS. This was due to the fact that, after sorting by FACS, protein

concentrations of candidate proteins were below the Western blot detection limit.

The β-galactosidase assay of three independent isolations revealed 60 %, 43 % and

46 % remaining β-galactosidase activity in TDM bead phagosomes compared to

control ones (results not shown). Figure 3.14 shows representative pictures of the

amount of proteins annexinA1, annexinA6, profilin1, SNAP23 and VAMP3 on control

versus TDM bead phagosomes. The bands below represent the loading control signal

for α-tubulin. Quantification of three independent experiments via densitometry

revealed that average annexinA1 was 35 %, annexinA6 75 %, profilin1 31 %, SNAP23

72 % and VAMP3 16 % more abundant on TDM versus control bead phagosomes.

Cofilin1 could not be detected.

Figure 3.14: Quantification of candidate proteins annexinA1, annexinA6, profilin1, SNAP23 and VAMP3 on isolated control versus TDM bead phagosomes via Western blot. Macrophages were “bead infected” with control and TDM beads with an MOI of 10. 30 mpi, bead phagosome maturation was stopped. Bead phagosomes were subsequently isolated and purified as described in chapter 2.2.1. Control and TDM bead phagosomes isolated and purified in three independent experiments were analyzed by Western blot and quantified by densitometry as described in 2.2.3. Figure 3.14 shows representative pictures of one experiment. Cofilin1 could not be detected by Western Blot. Average, annexinA1 was 35 %, annexinA6 75 %, profilin1 31 %, SNAP23 72 % and VAMP3 16 % more abundant on TDM versus control bead phagosomes.

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3.3.3 Evaluation of the role of candidate proteins for intracellular survival of M.

tuberculosis by small interfering RNA/CFUs analysis

Ultimately, the relevance of candidate proteins in early processes of M. tuberculosis-

mediated inhibition of phagosome maturation and consequently survival in

macrophages was tested. Therefore, candidate proteins were knocked-down in

RAW264.7 macrophages using the small interfering RNA (siRNA) technology followed

by infection with M. tuberculosis and analysis of colony-forming-units (CFUs) at 2 hpi,

4 hpi, 1 dpi, 2 dpi and 3 dpi (hours/days post infection). As control, untreated (negative

control, NK) and RAW264.7 macrophages transfected with non-targeting-RNA (NTR)

were used. Of note, due to technical difficulties only candidate proteins annexinA6,

cofilin1, profilin1 and VAMP3 could be analyzed.

The efficiency of siRNA-mediated knock-down of candidate proteins was tested via

Western blot. In this experimental set-up, the role of candidate proteins early in

infection should be tested. Thus only their absence on the day of infection was of

interest and evaluated by Western blot as shown in Figure 3.15. The upper bands

show the signals for annexinA6, profilin1, cofilin1 and VAMP3, 1 day (profilin1,

VAMP3) and 2 days (annexinA6, cofilin1) post transfection (dpt) with NTR and

targeting-siRNA in RAW264.7 macrophages, respectively. The bands below represent

the loading control signal for α-tubulin. Densitometry analysis revealed that, compared

to NTR controls, the amount of annexinA6 was reduced to 80 %, of cofilin1 to 5 %, of

profilin1 to 55 % and of VAMP3 to 2 %.

Figure 3.15: Control of siRNA-mediated knock-down of candidate proteins annexinA6, cofilin1, profilin1 and VAMP3 via Western blot. Macrophages were transfected with siRNA for annexinA6, profilin1, cofilin1 and VAMP3 and NTR as control. 1 dpt or 2 dpt, cells were infected with M. tuberculosis or harvested and destroyed by a freeze (N2 (l)) and thaw (37 °C) cycle. Samples were treated with DNAse and the protein concentration was determined using the Pierce 660 nm protein assay. Afterwards, 10 µg of protein were loaded onto a SDS-PAGE. Gels were blotted, blocked and incubated with antibodies against annexinA6, cofilin1, profilin1 and VAMP3 in

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parallel with an antibody against α-tubulin as loading control. Subsequently, blots were incubated with anti-rabbit coupled to HRP. Blots were washed, developed with an ECL-solution and exposed to Hyperfilm 10 sec to 2 min corresponding to band strength. Densitometry analysis revealed that, compared to NTR controls, the amount of annexinA6 was reduced to 80 %, of cofilin1 to 5 %, of profilin1 to 55 % and of VAMP3 to 2 % as assessed by Western blot.

Figure 3.15 shows the CFUs of M. tuberculosis from macrophages with reduced

amounts of (A) annexinA6, (B) cofilin1, (C) profilin1 and (E) VAMP3. Compared to NTR

controls, reduction of annexinA6 significantly lowered CFUs at 2 and 4 hpi. Similarly,

knock-down of cofilin1 revealed significantly decreased M. tuberculosis CFUs at 2 and

4 hpi and 1 dpi whereas reduced amounts of profilin1 decreased CFUs at 4 hpi and

1 dpi. Finally, the CFU of cells with reduced VAMP3 is significantly increased at 2 hpi.

Figure 3.16: Survival of M. tuberculosis inside RAW264.7 macrophages after knock-down of annexinA6, cofilin1, profilin1 and VAMP3 using RNA interference. Macrophages were transfected with NTR or siRNA for annexinA6, cofilin1, profilin1 and VAMP3. 1 dpi or 2 dpt, macrophages were infected with M. tuberculosis with an MOI of 0.5. As negative control, untreated macrophages were infected as well. CFUs were analyzed 2 hpi, 4 hpi, 1 dpi, 2 dpi and 3 dpi. The CFU assay was performed once with three technical replicates and analyzed with Two-Way-ANOVA with Bonferroni post test. NTR served as control for statistical analysis. n=3 technical replicates, p-values *< 0.5 *, < 0.01 **, < 0.001 ***.

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3.4 A role for the β-actin cytoskeleton in TDM-mediated inhibition

of phagosome maturation

A certain number of proteins identified in both control and bead phagosomes were

actin-interacting proteins including profilin1 and cofilin1. Additionally, actin was one of

the most abundant proteins identified with over 100 EUSC in both samples. (Table 0-1,

#571) Consequently, the localization of actin on control and TDM bead phagosomes

was examined.

Therefore, RAW264.7 macrophages were “bead infected” with control and TDM

beads. Bead phagosome biogenesis was stopped 30 min after phagocytosis and

macrophages were stained for β-actin using Phalloidin Alexa 488 (Figure 3.17).

Confocal fluorescence microscopy revealed that a certain percentage of TDM bead

phagosomes were decorated with β-actin (B) while control bead phagosomes were

devoid of β-actin (A). Quantitative analysis showed that circa 12 % of TDM bead

(arrows) but only 1 % of the control phagosomes were associated with β-actin rims at

30 min after phagocytosis (C).

Figure 3.17: Accumulation of β-actin around control and TDM bead phagosomes in RAW264.7 macrophages. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and macrophages were stained for β-actin using Phalloidin Alexa 488 (green). Bead phagosomes are indicated with *. β-actin accumulation around TDM bead phagosomes is marked with arrows. A & B: Representative pictures of control and TDM bead phagosomes in macrophages stained for β-actin, respectively C: For quantification, 100 control and TDM bead phagosomes from three independent experiments were counted and analyzed for the presence of β-actin. n=3 biological replicates, TTest, p-value * < 0.05, ** < 0.01, *** < 0.001.

Next, phagosomes of M. tuberculosis were analyzed for β-actin accumulation.

Therefore, RAW264.7 macrophages were infected with M. tuberculosis GFP and

stained for β-actin with Phalloidin Alexa 594. In contrast to the β-actin rims observed

on TDM bead phagosomes, the β-actin staining in M. tuberculosis GFP infected cells

revealed a punctate accumulation decorating the phagosome as shown in picture B

and D of Figure 3.18 (arrows).

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Figure 3.18: Localization of β-actin and WASH1 around M. tuberculosis GFP phagosomes in RAW264.7 macrophages. Macrophages were infected with M. tuberculosis GFP with a MOI of 3. 2 hpi, phagosome maturation was stopped and macrophages were stained for β-actin and WASH1 using Phalloidin Alexa 549 (red) and a primary antibody against WASH1 and secondary anti-rabbit IgG fluorescent antibody (white), respectively. β-actin and WASH1 accumulation around M. tuberculosis GFP phagosomes is marked with arrows. A: M. tuberculosis GFP. B: β-actin staining pattern in macrophages. C: WASH1 staining pattern in macrophages. D: merged picture.

Spontaneous polymerization of monomeric cytoplasmic actin is relatively inefficient.

Thus, polymerization is facilitated by several proteins acting in synergy. Actin

polymerization on target membranes is mediated by the Arp2/3 complex in cooperation

with nucleating-promoting-factors (NPFs). Recently, the WASH-complex was

discovered to be the major actin polymerisation-promoting complex on endosomes.

The WASH-complex is constituted by five proteins: strumpellin, SWIP, WASH1,

Fam21 and CCDC53. To analyze whether β-actin on phagosomes of M. tuberculosis

is associated with the WASH-complex, RAW264.7 macrophages infected with M.

tuberculosis GFP and stained for β-actin were co-stained for WASH1 (Figure 3.18).

Indeed, WASH1 co-localized with β-actin to the M. tuberculosis GFP phagosome as

indicated with arrows in pictures C and D [103].

To test whether the accumulation of β-actin around the phagosome of M. tuberculosis

is involved in inhibition of phagosome maturation, 30 min after M. tuberculosis GFP

infection, β-actin was removed with Latrunkulin A (LatA), a drug that depolymerises F-

actin. After 2 hpi and 4 hpi, phagosomes of M. tuberculosis GFP were analyzed for

their association with lysotracker (A) and the lysosomal marker LAMP1 (B) (Figure

3.19). Upon LatA treatment of RAW264.7 macrophages, the numbers of M.

tuberculosis GFP in mature phagosomes as indicated by lysotracker positive staining

were significantly increased at 2 hpi (A). In contrast, after 4 hpi LatA did not alter

lysotracker association with M. tuberculosis GFP. LAMP1 association with M.

tuberculosis was also significantly enhanced at 2 hpi but not 4 hpi (B).

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Figure 3.19: Co-localization of lysotracker and LAMP1 with M. tuberculosis GFP in RAW264.7 macrophages after removal of β-actin with LatrunkulinA (LatA). Macrophages were infected with M. tuberculosis GFP with an MOI of 0.5. 1 hpi, cells were treated with LatA for 30 min. 2 hpi and 4 hpi, infection was stopped and macrophages were stained for LAMP1 using a primary antibody against LAMP1 and secondary anti-rat IgG fluorescent antibody. 30 mpi macrophages were loaded with Lysotracker. The experiment was performed with three biological replicates. For quantification, 100 M. tuberculosis GFP phagosomes from three independent experiments were counted and analyzed for their association with Lysotracker and LAMP1. Statistical analysis was performed using a Two-Way-ANOVA with Bonferroni post test. n=3 biological replicates, p-value * < 0.05, ** < 0.01, *** < 0.001.

Removal of β-actin by LatA from M. tuberculosis GFP phagosomes in RAW264.7

macrophages was further analyzed for its influence on survival of M. tuberculosis.

Compared to controls (DMSO), the CFU in treated cells declined from 2 dpi to 3 dpi

on. At 6 dpi, the CFU was significantly decreased.

Figure 3.20: Survival of M. tuberculosis GFP in RAW264.7 macrophages after removal of β-actin with LatrunkulinA (LatA). Macrophages were infected with M. tuberculosis GFP with an MOI of 0.5. 1 hpi, cells were treated with LatA for 30 min. After 2 hpi, 4 hpi, 1 dpi, 2 dpi, 3 dpi and 6 dpi, CFUs were analyzed. The experiment is a representative one out of three. Statistical analysis was performed using a Two-Way-ANOVA with Bonferroni post test. n=3 technical replicates, p-value * < 0.05, ** < 0.01, *** < 0.001.

To determine whether WASH-complex mediated β-actin accumulation is important for

survival of M. tuberculosis phagosomes, WASH1 was knocked-down via small

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interference RNA in RAW264.7 macrophages. The efficiency of siRNA-mediated

knock-down of WASH1 was tested via Western blot. In this experimental set-up, the

role of the WASH-complex early in infection should be tested. Thus only its absence

on the day of infection was of interest. The upper bands show the amount of protein

of WASH1, 2 day post transfection (dpt) in RAW264.7 macrophages treated with NTR

and WASH1-targeting siRNA. The bands below represent loading control with α-

tubulin. Overall the amount of WASH1 was decreased successfully by siRNA knock-

down. Compared to 2 dpt NTR control, the amount of WASH1 was significantly

reduced to 0 % as assessed by Western blot.

Figure 3.21: Control of siRNA-mediated knock-down of WASH1 via Western blot. Macrophages were transfected with siRNA targeting WASH1 and NTR as control. 2 dpt, cells were infected with M. tuberculosis or harvested and destroyed by a freeze (N2 (l)) and thaw (37 °C) cycle. Samples were treated with DNAse and the protein concentration was determined using the Pierce 660 nm protein assay. Afterwards, 10 µg of protein were loaded onto a SDS-PAGE. Gels were blotted, blocked and incubated with antibodies against WASH1 in parallel with an antibody against α-tubulin as loading control ON at 4 °C. Subsequently, blots were incubated with anti-rabbit coupled to HRP. Blots were washed, developed with an ECL-solution and exposed to Hyperfilm 10 sec to 2 min corresponding to band strength. As assessed by Western blot, the amount of WASH1 could be reduced to 0 %.

2 dpt with WASH1-targeting siRNA, macrophages were infected with M. tuberculosis

and CFUs were analyzed 2 hpi, 4 hpi, 1 dpi, 2 dpi and 3 dpi. Over time, knock-down

of WASH1 had no major impact on CFUs when compared to controls although CFUs

were significantly increased compared to controls at 2 dpi.

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Figure 3.22: Survival of M. tuberculosis inside RAW264.7 macrophages after knock-down of WASH1 using RNA interference. Macrophages were transfected with NTR and siRNA targeting WASH1. 2 dpt, macrophages were infected with M. tuberculosis with an MOI of 0.5. As negative control, untreated macrophages were infected as well. CFU were analyzed 2 hpi, 4 hpi, 1 dpi, 2 dpi and 3 dpi. The CFU assay was performed once with three technical replicates and analyzed with Two-Way-ANOVA with Bonferroni post test. NTR served as control for statistical analysis. n=3 technical replicates, p-values *< 0.05 *, < 0.01 **, < 0.001 ***.

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3.5 The lipid coated bead phagosome lipidome

In order to analyze whether TDM affects the lipid composition of bead phagosomes,

RAW264.7 macrophages were “bead infected” with control and TDM beads.

Subsequently control and TDM bead phagosomes were isolated and purified, internal

standards were added and lipids were extracted. Samples were analyzed by mass

spectrometry for the lipid composition as described in 2.2.4. Comparison of control and

TDM bead phagosomes should then reveal TDM-mediated changes in the bead

phagosome lipidome.

Seven pairs of control and TDM bead phagosomes were compared. Prior mass

spectrometry analysis, the β-galactosidase activity of all pairs was determined. TDM

bead phagosome preparations 1 and 4 showed remaining β-galactosidase activities

of 55 %, 3 of 28 %, 2, 5, 6 and 7 of 62 %, 64 %, 69 % and 64 %, respectively.

Figure 3.23: β-galactosidase activity of all seven paired samples used for lipidomic analysis of bead phagosomes. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. β-galactosidase activity was determined as described in 2.2.1.7. Percentages are shown above bars.

We analysed the different classes of ceramides (Cer), cholesterol (Chol),

lysobisphosphatic acid (LBPA), lyso-phosphatidylcholine (LPC), phosphatidylcholine

(PC), plasmalogen phosphatidylcholine (PC-O), phosphatidylethanolamine (PE),

plasmalogen phosphatidylethanolamine (PE-O), phosphatidylglycerol (PG),

phosphatidylinositol (PI), phosphatidylserine (PS) and sphingomyelins (SM).

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For a first general overview about the lipid composition of bead phagosomes, Figure

3.24 shows the lipid class composition of matured control bead phagosomes.

Accordingly, with 24 %, 23 % and 19 %, PC, LBPA and cholesterol were the most

prominent lipids in the bead phagosome lipidome, respectively, followed by SM (9 %),

PC-O (6 %), PI (5 %), PS (5 %), PE-O (4 %), PE (3%), Cer (1 %), PG (1 %) and LPC

(0.3 %).

Figure 3.24: Identified lipid classes in control bead phagosomes. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. Subsequently, internal standards were added, lipids were extracted and pairs of control and TDM bead phagosomes samples were analyzed for their lipid content by mass spectrometry as described in 2.2.4. Results were processed using Microsoft Excel and Prism 6.0. The pie chart shows the composition of lipid classes identified in matured control bead phagosomes.

The lipid profile was compared between control and TDM bead phagosomes.

Therefore, mol % of lipid classes of matching pairs were analyzed with a paired TTest.

Figure 3.25 and Figure 3.26, show the befor-and-after-plot of the 12 lipid classes

identified in all seven control (before) and TDM (after) bead phagosome pairs. Overall,

the lipid composition of control and TDM bead phagosomes was comparable.

However, three lipid classes were significantly different in TDM samples compared to

controls. While amounts of LBPA (Figure 3.25, C) were lower in TDM samples

compared to controls, amounts of cholesterol (Figure 3.25, B) and PS (Figure 3.26, E)

were significantly enhanced.

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Figure 3.25: Before-and-after-plot of Cer, Chol, LBPA, LPC, PC and PC-O identified in control versus TDM bead phagosomes. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. Subsequently, internal standards were added, lipids were extracted and pairs of control and TDM bead phagosomes samples were analyzed for their lipid content by mass spectrometry as described in 2.2.4. Results were processed using Microsoft Excel and Prism 6.0. Statistical analysis was performed with a paired TTest. n=7 biological replicates, p-value * < 0.05, ** < 0.01, *** < 0.001.

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Figure 3.26: Before-and-after-plot of PE, PE-O, PG, PI, PS and SM identified in control versus TDM bead phagosomes. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. Subsequently, internal standards were added, lipids were extracted and pairs of control and TDM bead phagosomes samples were analyzed for their lipid content by mass spectrometry as described in 2.2.4. Results were processed using Microsoft Excel and Prism 6.0. Statistical analysis was performed with a paired TTest. n=7 biological replicates, p-value * < 0.05, ** < 0.01, *** < 0.001.

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The TDM-induced changes in lipid class composition of bead phagosomes were

further analyzed on lipid species level. Summarizing, 16 lipid species were identified

for ceramides, 1 for cholesterol, 48 for LBPA, 5 for LPC, 38 for PC, 34 for PC-O, 30

for PE, 38 for PE-O, 31 for PG, 23 for PI, 27 for PS and 21 for SM (Table 0-2)

For graphic representation of single lipid species, a volcano-plot was chosen (Figure

3.27). Therefore the mean of p-values calculated from the mol % of all identified lipid

species in sample pairs was transformed to a negative log10 value and plotted against

the mean of the mol % fold change (ratio of TDM/control) of all sample pairs

transformed to a log2 value. This gives a volcano-plot as shown in Figure 3.27. Every

spot represents a single lipid species. Single lipid species shift left when decreased

and right when increased in TDM samples compared to controls. If single lipid species

have a paired p-value <0.05 and thus are significantly altered, they have a -log10 value

>1.13 (dashed horizontal line). If the fold change is >1.5 or >2, log2 values are >0.58

or >1 (dashed red vertical lines), respectively. If the fold change is <0.75 or <0.5, log2

values are <-0.41 or <-1 (dashed green vertical lines), respectively. Lipid species that

center around 0 are equally distributed in control versus TDM bead phagosome

samples.

The volcano-plot confirms the decrease of LBPA (turquoise dots) because most of the

LBPA species shift to the left. Species 44:10 but most prominently, species 36:2 is

significantly reduced in TDM bead compared to control phagosomes. Similarly, the

increase in PS in TDM samples compared to controls is revealed by the shift of several

PS (dark green) lipid species to the right. PS 37:1, PS 38:4 and PS 38:5 are

significantly accumulated in TDM samples compared to controls. The single light green

spot marked with “Chol” indicates a minor but significant increase in TDM bead

phagosome samples. Although increased without statistical significance on the lipid

class level, ceramides 32:1, 34:0, 34:1 and 41:2 are significantly accumulated in TDM

bead phagosomes. Similarly, SM species 40:1, 41:1, 42:2 are significantly increased

in TDM bead phagosomes.

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Figure 3.27: Volcano-plot - Distribution of identified lipid species in control versus TDM bead phagosome samples. Macrophages were “bead infected” with control and TDM beads with a MOI of 10. 30 mpi, bead phagosome maturation was stopped and bead phagosomes were isolated and purified as described in 2.2.1. Subsequently, internal standards were added, lipids were extracted and pairs of control and TDM bead phagosomes samples were analyzed for their lipid content by mass spectrometry as described in 2.2.4. For volcano-plot graphic representation, -log10 transformed p-values and log2 transformed fold changes of control versus TDM sample pairs were plotted. Every dot represents a single lipid species. Unchanged lipid species centre around 0. Altered lipid species shift left when decreased and right when increased in TDM bead phagosomes. Significant changes (p-value <0.05) appear above the horizontal black dashed line. Fold-changes >1.5 or >2 appear beyond red vertical dashed lines and fold-changes <0.75 or <0.5 appear beyond the green vertical dashed lines. Results were processed using Microsoft Excel and Prism 6.0. n=7 biological replicates

Discussion

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4 Discussion

4.1 Resume

Localized in the mycobacterial outer membrane, the cell-wall lipid TDM is a major

virulence factor of M. tuberculosis. When coated to beads, TDM was shown to inhibit

bead phagosome maturation [90], [91]. However, the molecular function underlying

TDM-mediated inhibition of phagosome maturation remains elusive. We hypothesized,

that TDM exerts its virulence function by interacting with host-cell partners at the

phagosome interface. In the TDM determined vesicle, these interaction partners are

present either within the phagosome, inside the phagosomal membrane or linked to

the cytoplasmic side of the phagosome. The direct or indirect interaction of TDM with

these interactors putatively affects phagosome maturation. Thus the main aim of this

thesis was to identify TDM direct or indirect interaction partners. To study the effect of

TDM, a simplified lipid-coated bead model was used. Beads were coated with TDM

and used for “bead infection” of macrophages to isolate and purify bead phagosomes

for mass spectrometry based proteomics and lipidomics.

To identify and quantify potential interaction partners, a protocol had to be developed

that enabled isolation and purification of bead phagosomes for mass spectrometry

analysis. An existing protocol including (i) coating of beads with TDM, (ii) “bead

infection” of macrophages (iii) bead phagosome isolation from macrophages by

magnet and (iv) purification of bead phagosomes by DNase digestion and Ficoll-

gradient centrifugation was refined by addition of a protease digestion step and sorting

bead phagosomes by FACS.

Proteomics of control and TDM bead phagosomes identified 1054 proteins in total.

Grouping of identified proteins according to their cellular localization revealed 2916

proteins that could be assigned as phagosome-associated whereas 898 proteins were

potential contaminants indicating an enrichment of phagosomal proteins. After

application of exclusion criteria, 34 proteins were filtered that were enriched in TDM

bead phagosomes compared to controls and thus potential TDM interaction partners.

Of these, annexinA1, annexinA6, cofilin1, profilin1, SNAP23 and VAMP3 were chosen

for further analysis because they have been described to be associated with

phagosomes as part of a phagosome-interacting network.

Discussion

96

Immunofluorescence to verify mass spectrometry results, however failed to reveal

specific association with TDM bead or M. tuberculosis phagosomes. Notably, cofilin1

and profilin1 were found positive to LAMP1 M. tuberculosis phagosomes. In contrast

to the immunofluorescence approach, Western blot analysis of isolated and purified

bead phagosomes confirmed enrichment of all 6 candidate proteins on TDM bead

phagosomes compared to control ones. Preliminary siRNA experiments to knock-

down candidate proteins in macrophages for subsequent infection with M. tuberculosis

indicated that absence of annexinA6 and VAMP3 impairs phagocytosis and that

cofilin1 and profilin1 are important for survival of the pathogen in macrophages.

Furthermore, proteomics revealed enrichment of β-actin and β-actin-binding-proteins

in bead phagosome proteomes. Fluorescence staining using Phalloidin demonstrated

accumulation of β-actin on TDM bead and a punctate pattern on M. tuberculosis

phagosomes that co-localized with NPF WASH1. Removal of β-actin drove M.

tuberculosis in phago-lysosomes and impaired survival of M. tuberculosis in

macrophages. However, preliminary RNA interference experiments suggested that

WASH1 is dispensable for survival of M. tuberculosis in macrophages.

Differential lipidomics by mass spectrometry of control and TDM bead phagosomes

demonstrated that TDM bead phagosomes had significantly less LBPA and

significantly increased amounts of cholesterol and phosphatidylserine compared to

control ones. Analysis at the single lipid species level additionally revealed significant

increase of certain ceramide and sphingomyeline species in TDM bead phagosomes

when compared to control ones.

In conclusion, these results reflect that TDM and control bead phagosomes vary on

protein and lipid levels because each bead phagosome represents a different

maturation stage. However, identified TDM interactors may have an important direct

or indirect functional role in inhibition of phagosome maturation. Notably, there is a

specific association of the actin cytoskeleton network with non-maturing M.

tuberculosis and TDM bead phagosomes. A model for actin cytoskeleton function in

interference with phagosome biogenesis by TDM is proposed.

Discussion

97

4.2 Method development: Isolation and purification of bead

phagosomes from macrophages for mass spectrometry

analysis

Phagocytosis and subsequent phagosome maturation is a hallmark of innate immune

cells. The process of phagosome maturation is designed to ingest and eliminate

engulfed particles by a well orchestrated mechanism (Figure 1.5). Phagocytosis is not

only important for elimination of pathogens but links innate and acquired immunity

through antigen processing and presentation to T-cells. Nevertheless, several

intracellular pathogens have developed distinct strategies to interfere with phagosome

maturation and thus survive and thrive within their niche. Due to the fact that the

successful manipulation of phagocytosis and phagosome maturation determines the

outcome of infections, the underlying intracellular events are of outermost importance

for novel concepts of infection control.

In the past decades, several techniques for isolation and purification of phagosomes

were developed. These techniques underlie three different principles: (i) choosing the

host-cell and the phagocytic particle, (ii) infection, destruction and isolation of the

phagosome from host-cells and (iii) purification of phagosomes. Current methods are

mainly based on experiments performed by Wetzel and Korn in 1969 [105]. In principle,

polystyrene latex-beads were fed to amoeba, isolated by a Potter-Elvehjem-

homogenizer and purified by density gradient centrifugation using a continuous

sucrose gradient. Latex bead phagosomes (LBPs) can be prepared with high purity

because of their significantly distinct density compared to host-cell organelles.

In the following years, similar separation protocols have been applied to isolate and

purify LBPs from murine and human macrophages [106],[107] and amoeba

[108],[109]. Density gradient centrifugation was also applied to isolate pathogen-

containing vacuoles (PCVs) from macrophages [98], [110]–[112]. Nevertheless,

because PCVs have densities closer to certain organelles, multiple steps of purification

by differential density centrifugation are typically employed [113]. To circumvent the

time-consuming ultra-centrifugation steps, magnetic particles were introduced in the

field of phagosome cell biology [114]. In these protocols, both magnetic beads or

viable, magnetically labelled pathogens such as BCG or M. tuberculosis were studied

[115]–[117]. For phagosome isolation, whole cell homogenates containing

phagosomes were subjected to a strong magnetic field and further purified by several

Discussion

98

washing steps [114]–[117]. Upon removal of the magnet, the phagosomes were

collected and further enriched. Furthermore, organelle and free-flow electrophoresis

were used for purification of PCVs. Here PVCs were separated from other intracellular

organelles in homogenates of infected macrophages by electrophoresis through a

gradient according to their charge and size [111]. However, using the described

methods, phagosomes could never be perfectly purified since complete separation

from other cell constituents was never achieved due to the complex interaction of

phagosomes with other compartments in the cytoplasm [113]. Consequently,

phagosomes were rather enriched than purified.

In the present study, magnetic bead phagosomes were isolated and purified from

macrophages. The practical procedure included (i) bead infection of macrophages with

magnetic beads, (ii) homogenization of macrophages using a douncer, (iii) isolation of

magnetic bead phagosomes with a magnet and (iv) purification of bead phagosomes

by DNase and trypsin digestion steps, Ficoll-gradient centrifugation and final sorting

by FACS.

Magnetic (Dyna-) beads are polystyrene beads with enclosed magnetic particles. The

bead surface is tosylactivated to covalently bind proteins/peptides via primary amino-

or sulfhydryl groups. We covalently bound BSA to link TDM to the magnetic beads.

From previous experiments, we knew that this technique is suitable to coat beads with

TDM and that these TDM beads are able to inhibit phagosome maturation [91]. Binding

is most likely enabled by hydrophobic interactions between the mycolic acids of TDM

and hydrophobic pockets of BSA. Accordingly, the trehalose-moiety of TDM most likely

sticks out for interaction with host-cell “targets”.

Disruption of macrophages to isolate bead phagosomes is a delicate task since

phagosomes must remain intact. In many protocols for isolation of LBPs,

homogenization is achieved by passing cells through syringes with needle gauges

ranging from 25 to 28 [113]. This worked fine for LBPs since they have a much smaller

diameter (0.8-1.1 µm). We homogenized macrophages with a douncer. This method

allows destruction of macrophages without affecting phagosome integrity since the

gap between the pestle and the douncer chasses (13 µm) is wide enough for bead

phagosomes (Ø 2.8 µm) to stay intact.

Digestion of DNA with DNase is a very important step in the process of phagosome

purification because, due to their negative charge attracting cell debris, nucleotides

Discussion

99

(DNA in particular) can change a sample into a sticky gel impeding all further

purification efforts [98].

We noticed that, despite removal of DNA, bead phagosomes still stuck together,

probably due to protein-meshworks linking bead phagosomes together. Thus samples

were additionally treated with trypsin. Protease digestion was applied for phagosome

isolation procedures before [111]. However, it is still an unusual step, due to the fact

that proteases act non-targeted and therefore may also digest proteins of interest.

Thus, this step had to be performed very precise regarding incubation time and sample

protein to trypsin concentration and the enzyme had to be effectively removed

afterwards. However, maintained β-galactosidase activity in phagosome preparations

after trypsin treatment indicated bead phagosome integrity.

Sucrose gradient centrifugation takes advantage of the fact that latex beads have a

relatively low density and thus migrate in sucrose gradients differently from other cell

organelles [105]. Polystyrene beads including magnetic ones used herein have a

relatively high density, which precluded sucrose gradients from being applied for

further purification. Therefore, we used a Ficoll-gradient to separate phagosomes from

cellular debris. Ficoll-gradients are hydrophilic polysaccharide meshworks commonly

used to separate cell populations but also allowed us to separate magnetic bead

phagosomes from cellular debris and other organelles [98], [115], [118].

However, our own results and other reports on isolation of particle containing

phagosomes showed that contaminations with non-phagosomal host-cell material

from distinct compartments such as ER, mitochondrial or nucleus cannot be

completely avoided by the purification steps described above. Together with

increasing sensitivity of mass spectrometry methods in recent years, purity of

phagosome preparations is a significant bottleneck because high abundant

contaminating proteins can overwrite signals of phagosomal proteins with lower

abundance [119]. In 1998, Ramachandra et al. discussed sorting by flow cytometry as

an easy and rapid method for isolating phagosomes from cell lysates [120]. Sorting by

FACS was applied to isolate Salmonella and Staphylococcus aureus phagosomes

from infected cells [121]–[123]. We introduced FACS sorting as a new method to

further purify bead phagosomes. Bead phagosomes were sorted by size, re-isolated

using a magnet and analyzed by mass spectrometry. Proteins in the supernatant from

phagosomes which were destroyed during sampling into tubes after sorting were

precipitated using protein-binding beads [124]. Mass spectrometry analysis revealed

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100

that the majority (74 %) of identified proteins were present in bead phagosome

samples whereas only minor (26 %) amounts of proteins were found in the supernatant

precipitates. Thus, we are confident that most bead phagosomes remained intact

during sorting by FACS. Analysis of the cellular localization of identified proteins

showed strong accumulation of endosomal/phagosomal proteins (2916) compared to

non-intracellular vesicle proteins (898).

Taken together, the newly developed protocol is suitable for purification of bead

phagosomes for subsequent mass spectrometry analysis (Figure 3.1).

4.2.1 Quantification of phagosomal proteins by spectral counting is a label-free

and semi-quantitative approach

In the present work, quantification of proteins was accomplished by spectral counting.

According to the Scaffold 4 software used for evaluation of data, the EUSC (exclusive

unique spectrum count) describes the number of spectra that can be assigned to only

one specific peptide/protein and does not fit other peptides/proteins. Quantification by

spectral counting is classified as semi-quantitative because it is a label-free method.

Spectral counting is based on the empirical observation that the more of a particular

protein is present in a sample, the more tandem MS-spectra are collected for peptides

of the same protein [125]. In contrast, with label-based methods, mass spectrometry

can recognize mass differences between the labelled and unlabeled forms of a peptide

and quantification is achieved by comparing their respective signal intensities [125].

Quantification by spectral counting has several disadvantages. First, the spectrum

count response for every peptide is different since e.g. the chromatographic behaviour

(retention time, peak width) varies. Furthermore, at higher spectral counts, saturation

effects will occur which are different for every peptide [125]. In addition, measurement

accuracy of the mass spectrometer varies prone to measurement errors.

Nevertheless, the correlation between amount of protein and number of tandem mass

spectra yields reliable results and quantification by spectral counting is applied

routinely in proteomics [126].

4.2.2 Evaluation of proteomics data by immunofluorescence staining and

Western blot revealed contradicting results

In the present study, proteomics data of isolated control and TDM bead phagosomes

were evaluated by IF-staining and Western blot. For Western blot experiments, the

isolation and purification protocol did not include sorting by FACS. We excluded this

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101

purification step, because protein concentration of samples (107 bead phagosomes)

after sorting by FACS was below the Western blot detection limit.

Evaluation of proteomics data by IF-staining and Western blot, gave very different

results. While Western blot experiments confirmed proteomics data, i.e. enrichment of

candidate proteins in TDM bead phagosomes, the respective proteins could not be

detected on bead phagosomes by IF-staining. These results may be due to the

epitopes of the antigens recognized by the antibodies in their native (IF-staining)

versus linearized (Western blot) form. However, the staining pattern of all candidate

proteins in macrophages resembled that shown in previous reports of those

antibodies. Another possibility, the antigens could be present in a different

conformation at phagosomes compared to other compartments.

Discussion

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4.3 TDM induced changes on the bead phagosome proteome

A “proteome” is described as the complement of proteins in a sample as e.g. isolated

phagosomes. Up to date, searching “phagosome” and “proteome” on pubmed.com

revealed 68 hits. Probably one third of these results covered LBP studies, the gold

standard to study phagocytosis and phagosome maturation. Only one third dealt with

PCV-proteomes. Searching “Mycobacterium tuberculosis”, “phagosome” and

“proteome” only gave 5 results. Although analysis of isolated mycobacterial

phagosomes is reported several times, only 3 studies presented whole proteomes of

Mycobacteria containing phagosomes.

Mycobacterial phagosomes have been characterized before by various approaches

[119], [131], [132]. Due to the fact that Mycobacteria maintain early endosomal

characteristics, they acquire proteins of the early and intermediate phagosomes stage

such as the transferrin receptor (TfR), Rab5, EEA1, synthaxin4, synthaxin6 and

VAMP3 and carry limited amounts of v-H+-ATPase, lysosomal hydrolases including

cathepsinD, LAMP1/2 and Rab7 [129]–[131]. In a previous study with TDM beads, IF-

staining and Western blot experiments confirmed retention of TfR and exclusion of

LAMP1 and mature cathepsinD [91].

Our proteomics data corroborate that TfR and VAMP3 are enriched on TDM bead

phagosomes compared to controls (# 303, 166, Table 0-1). Rab5a and Rab5c were

equally distributed in both samples (# 208, 456, Table 0-1). Syntaxin4 was only found

in the TDM sample (#128, Table 0-1). In contrast, syntaxin6, EEA1 and LAMP2 were

not detected. In both samples, the v-H+-ATPase was one of the most abundant

proteins since plenty subunits were identified (# 16, 200, 262, 380, 389, 416, 426, 445-

447, 837, 1020, Table 0-1). Nevertheless, lower abundance of the v-H+-ATPase on

TDM bead phagosomes was not observed. In fact, many subunits were accumulated

in TDM samples. Levels of LAMP1 were almost similar in both samples with a trend to

lower amounts in TDM samples (# 612, Table 0-1). In contrast, Rab7a was strongly

accumulated at TDM bead phagosomes compared to controls (#233, Table 0-1).

These data indicate that TDM bead phagosomes share characteristics with M.

tuberculosis phagosomes but differ regarding other ones.

In the present study, 843 proteins were identified in TDM bead phagosomes.

Proteomic analysis of BCG containing phagosomes from the human monocyte cell line

THP-1 identified 447 proteins [127]. Similar to our results, proteins from several

Discussion

103

categories were identified. Table 4-1 provides a coarse overview about proteins

identified in BCG versus TDM bead phagosomes:

Table 4-1: Comparison of proteins identified in BCG versus TDM bead phagosomes. Modified from [127]. The left column provides a coarse overview of proteins identified in BCG phagosomes. The right column lists proteins identified in BCG phagosomes that were present in TDM phagosomes as well. Underlined proteins were exclusively present in BCG compared to latex bead phagosomes.

BCG phagosome proteome TDM bead phagosome proteome

Endosomal/lysosomal proteins:

CD63, LAMP1, LAMP2, lysosomal membrane protein 2, subunit B of v-ATPase, cathepsin D, cathepsin Z, palmitoyl-protein thioesterase, lysosomal acid phosphatase, N-acetylglucosamine-6-sulfatase, lysosomal α-glucosidase, acid ceramidase, prosaposin, M6PR, Golgi apparatus protein 1, mannosidase-2

LAMP1, subunit B of v-ATPase, cathepsin D cathepsin Z, palmitoyl-protein thioesterase, acid ceramidase, M6PR, mannosidase-2

Multivesicular body and exosome pathway:

Alix, CHMP4 integrin β1/2 / α-5, CD63, Rap-1B/1A, Rab7, annexins A2, A4, A5, Hsc70, Hsp90, actin, cofilin, tubulin, tubulin α-1C chain / β-chain, moesin, pyruvate kinase, enolase, elongation factor 1-α/14-3-3, stress-induced-phosphoprotein 1, charged multivesicular body protein 4b, septin-7, sec22b

Alix, CHMP4 integrin β1/2 / α-5, Rap-1B/1A, Rab7, annexins A2, A4, A5, actin, cofilin, tubulin, moesin, enolase, elongation factor 1-α/14-3-3, septin-7, stress-induced-phosphoprotein 1

Lipid raft proteins:

flotilin1, stomatin, erlin2

stomatin

Signaling proteins:

CD44, ADP-ribosylation factor (Arf)-like protein 8b/8a, brain acid-soluble protein 1, heterotrimeric G-proteins GNAI2, GNAI3, GNB1, GNG5, GNG12, IQGAP1, 14-3-3 family proteins β/α, ε,η,γ, ζ/δ and θ, CAP1, very long-chain-specific acyl-CoA dehydrogenase, ADP/ATP translocase 2, guanine nucleotide-binding protein Gi/Gs/Gt subunit β-1, platelet endothelial cell adhesion molecule

CD44, brain acid-soluble protein 1, 14-3-3 family proteins β/α, ε,η,γ, ζ/δ and θ, CAP1, platelet endothelial cell adhesion molecule

Discussion

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Rab and Rab-related proteins:

Rab7a, Rab11a, Rab11b, Rab1a, Rab1b Rab6a, Rab21, Rab14, RalB, RalA, Rap1A/1B, GDI, Ras GTPase-activating-like protein IQGAP1

Rab7a, Rab11A, Rab21, Rab14, Rab6a

Secreted proteins:

Apolipoprotein D/E, complement factors C3/C9, retinoid-inducible serine carboxy-peptidase

retinoid-inducible serine carboxy-peptidase

Proteins of unknown function:

Fam3C, tumor protein D54, VAT1, transmembrane emp24 domain-containing protein 9

Fam3C, tumor protein D54

Others:

glutamate dehydrogenase 1, nucleobindin-1, vacuolar proton pump subunit C1, 60 S ribosomal protein L35, 60 S ribosomal protein L8, macrophage migration-inhibitory factor, translocon-associated protein subunit α, band 4.1-like protein 3, intracellular adhesion molecule 1

vacuolar proton pump subunit C1, 60 S ribosomal protein L35, 60 S ribosomal protein L8, macrophage migration-inhibitory factor, translocon-associated protein subunit α, band 4.1-like protein 3, intracellular adhesion molecule 1

In the study by Lee et al., BCG phagosomes were further compared to LBPs to detect

BCG phagosome specific proteins revealing 32 exclusive proteins in BCG

phagosomes (underlined in Table 4-1). Of these, 17 were not present in TDM bead

phagosomes indicating specificity for BCG. Overall, comparison with our data revealed

a large overlap between the TDM and BCG phagosome proteomes.

In another study performed by Rao et al., proteomes of phagosomes with either M.

tuberculosis H37Rv, attenuated strain M. tuberculosis H37Ra or BCG were compared

in a systems biology approach [132]. Principal component analysis revealed that

proteins Hsd17b12, Rpl38, Rpl6, nicastrin, Rab11b, Gnb1, Rps3a, Cdc42 and

C230096C10Rik were specifically enriched in M. tuberculosis H37Rv phagosomes. Of

these, only Hsd17b12 and Gnb1 were found in TDM bead preparations indicating

exclusive enrichment in phagosomes containing viable M. tuberculosis. In contrast,

the of proteins Cct8, Asns, Nomo1, Ptcd3, Rpl26, Rpl11, Cyb5b and Rab5a enriched

in BCG phagosomes, only Ptcd3, Rpl26 and Rpl11 were absent in TDM bead samples.

Discussion

105

This comparison indicates that TDM bead phagosomes may share more

characteristics with phagosomes containing BCG than those with M. tuberculosis. Of

note, the whole phagosome proteome data of M. tuberculosis H37Rv and BCG were

not available for comparison to the scientific community.

In experiments similar to ours, Shui et al. coated latex-beads with ManLAM from M.

tuberculosis, PILAM (phosphoinositol-capped LAM) from M. smegmatis as well as LPS

from E.coli [133]. All three bead types were fed to RAW264.7 macrophages and the

membrane fraction of bead phagosomes was isolated and analyzed in an iTRAQ-

labelled approach by mass spectrometry. 823 proteins were identified in total and 42

proteins were significantly up- or down-regulated (p > 1.25-fold, p < 0.05-fold) by

exposure of macrophages to ManLAM but not the other two lipoglycan beads:

Table 4-2: Comparison of proteins identified in ManLAM versus TDM bead phagosomes. Modified from [133]. The left column provides the list of proteins up- and down-regulated in ManLAM bead phagosomes compared to controls. The right column lists proteins identified in ManLAM bead that were present in TDM bead phagosomes as well.

ManLAM-specific phagosome proteome

TDM bead phagosome proteome

up-regulated:

transferrin receptor protein 1 vacuolar protein sorting-associated

protein 41 homologue Rab-5A Rab-5C Rab-14 isoform 1 of sequestosome-1 isoform 2 of sequestosome-1 ferritin H subunit calnexin heat-shock protein 90B1 tripeptidyl-peptidase 1 N-acetylglucosamine-6-sulfatase D-3-phosphoglycerate dehydrogenase alpha-N-acetylglucosaminidase vimentin titin 24 kDa protein Ddost

transferrin receptor protein 1 Rab-5A Rab-5C Rab-14 Calnexin tripeptidyl-peptidase 1 D-3-phosphoglycerate dehydrogenase alpha-N-acetylglucosaminidase Ddost

down-regulated:

mannose-6-phosphate receptor CD63 scavenger receptor class B member 2 lysosomal membrane glycoprotein 1 lysosomal membrane glycoprotein 2 transmembrane protein 55A

mannose-6-phosphate receptor lysosomal membrane glycoprotein 1 transmembrane protein 55A

Discussion

106

early endosome antigen 1 vesicle transport through interaction with

t-SNAREs homologue 1B Rab-7A Rab-7B immune regulator 1, ATPase, H+

transporting ATPase, H+ transporting, lysosomal V0

subunit a vacuolar ATP synthase subunit B vacuolar ATP synthase subunit E1 vacuolar ATP synthase subunit S1 vacuolar ATP synthase subunit C vacuolar ATP synthase subunit F vacuolar ATP synthase subunit d vacuolar ATP synthase catalytic subunit

A isoform 1 of reticulon-4 cathepsin Z lysosomal acid phosphatase collectin subfamily member 12 zinc finger, FYVE domain containing 26

vesicle transport through interaction with

t-SNAREs homologue 1B Rab-7A ATPase, H+ transporting, lysosomal V0

subunit a vacuolar ATP synthase subunit B vacuolar ATP synthase subunit d isoform 1 of Reticulon-4 cathepsin Z

Of the 18 proteins up-regulated in ManLAM bead phagosomes, 9 were present in TDM

bead phagosomes and of these the TfR1, Rab14, calnexin and the tripeptidyl-

peptidase 1 were also within the group of proteins enriched in TDM bead phagosomes.

Of the 24 proteins down-regulated in ManLAM bead phagosomes, 9 were present in

TDM samples and similarly, LAMP1 and the v-type ATPase subunit b were decreased

compared to controls. This comparison indicates that phagosomal proteomes of

ManLAM and TDM beads share certain proteins. However, proteins identified in both

samples were differently regulated.

To summarize, most of the early/intermediate phagosomal marker proteins found on

mycobacterial phagosomes were also found enriched in TDM bead phagosomes

compared to controls. Similarly, late phagosomal marker proteins were absent or

decreased on TDM bead phagosomes compared to controls. In addition, comparison

with phagosomes from BCG, M. tuberculosis and ManLAM coated LBs, revealed an

overlap of proteins also identified in TDM bead phagosomes. These results further

validate the purification method applied as suitable to achieve sufficiently pure bead

phagosomes from macrophages.

Discussion

107

4.3.1 TDM bead phagosomes accumulate proteins of the annexin superfamily

In vertebrates, the annexin superfamily encompasses twelve members (A1-11, A13).

Annexins are best characterized by their ability to bind negatively charged

phospholipids in a Ca2+-dependant manner as they are composed of two domains: a

conserved core that is responsible for Ca2+- and phospholipid-binding and a variable

N- terminal tail. This property links annexins to many membrane-related events, such

as the regulated organization of membrane domains and/or membrane–cytoskeleton

linkages and certain exocytic and endocytic transport steps [134].

Evaluation of control versus TDM bead phagosomes revealed that two members of

the annexin superfamily, namely annexin A1 and A6, were significantly enriched on

TDM bead phagosomes and thus potential direct or indirect TDM interaction partners.

These results confirmed previous findings demonstrating annexin A1 and A6 on

endosomal compartments [134]. Recent studies also identified both proteins on

phagosomes of Mycobacterium avium [102]. Verification of the presence of annexinA1

and A6 on isolated and purified control and TDM bead phagosomes by Western blot

experiments corroborated proteomics data of accumulation to TDM bead samples.

Preliminary experiments using small interference RNA-mediated knock-down in

macrophages infected with M. tuberculosis suggested that annexinA6 affected

phagocytosis but not survival of the pathogen.

Functional relevance of annexinA1 for phagosome biogenesis has been established

as it was identified on phagosomes containing yeast, E. coli, dead Brucella suis [135],

opsonized zymosan [136] or promastigote Leishmania mexicana [137]. Furthermore

macrophages from annexinA1-knockout mice showed impaired phagocytosis and

knockout of annexinA1 has been shown to alter cytokine production and phagocytic

receptor expression in mouse macrophages [134]. Nevertheless, function of

annexinA1 on phagosomes remains largely unknown although in vitro data suggest a

link to the actin cytoskeleton as annexinA1 binds to and bundles F-actin in vitro, co-

localises with F-actin in different cell lines and interacts with profilin1 [138],[139].

Recently, Patel et al. could show that annexinA1 (i) facilitated phagosome-F-actin

interaction in an ATP-independent manner in vitro, (ii) co-localised with F-actin during

phagocytic cup formation and on mature phagosomes in vivo and (iii) that the presence

of annexinA1 correlated with the time-dependent association of F-actin with

phagosomes. Moreover Patel el al. revealed that knock-down of annexinA1 impaired

phagocytosis of latex-beads. All together, these results strongly suggest a role for

Discussion

108

annexinA1 in controlling association of actin with phagosomes during phagocytosis

but also during phagosome biogenesis.

Association of annexinA6 to phagosomes was initially demonstrated for latex bead

phagosomes by Desjardins and co-workers in 1994 [140]. In the following years,

annexinA6 was detected on early and late endosomal membranes as well [134]. In

contrast to other annexins, annexinA6 has two core-modules and thus allows the

molecule to bind to either 1 membrane or 2 separate ones. AnnexinA6 binds negative

charged phospholipids such as PS, PI, PA, PE and arachidonic acid in a Ca2+-

dependent manner [141]. Moreover annexinA6 was revealed to bind cholesterol in the

absence of Ca2+ [141]. Mass spectrometry analysis of the lipid composition of bead

phagosome membranes revealed that PS and cholesterol were accumulated in TDM

bead phagosomes compared to controls (see below). As PS and cholesterol are

preferred binding partners of annexinA6, both lipids may have contributed to

enrichment of annexinA6 to TDM bead phagosomes [142].

AnnexinA6 was described to modulate intracellular cholesterol homeostasis, as its up-

regulation resulted in cholesterol accumulation in late endosomes and consequently

inhibited cholesterol export to the Golgi apparatus and plasma membrane [141].

Similar to annexinA1, A6 was also demonstrated to regulate membrane-actin

interactions during endo- and exocytosis [141]. Enrich et al. recently demonstrated that

membrane localization of annexinA6 caused a rearrangement of F-actin at the plasma

membrane stabilizing the cortical actin cytoskeleton. Besides, interacting with F-actin,

annexinA6 was identified as a binding partner of spectrin. Furthermore, annexinA6

regulating receptor-mediated endocytosis via spectrin was proposed to be involved in

the internalization of LDL (low density lipoprotein) [143].

Enrichment of both annexins to TDM bead phagosomes could simply reflect their early

phagosomal stage as annexinA1 and A6 were shown to be important for actin

assembly during phagocytosis or receptor-mediated endocytosis, respectively. On the

other hand, due to their preferential binding to lipids such as PS and cholesterol

accumulated to TDM phagosomes, annexinA1 and A6 could be attracted by TDM via

phagosomal membrane lipids to function as actin nucleation-promoting-factors (NPFs)

or to attract additional NPFs. This may promote formation of actin rims, which can limit

phagosome maturation (Figure 3.17). The latter hypothesis is further supported by the

partially overlapping intracellular localization of annexinA1 and profilin1 and annexinA6

and spectrin [139], [141]. Both, profilin1 and spectrin are known to regulate actin

Discussion

109

nucleation. One could speculate that the annexinA1-profilin and annexinA6-spectrin

interaction participates in regulating the membrane-associated actin cytoskeleton

[143]. However, further evaluations of these protein interactions are required.

4.3.2 TDM bead phagosomes accumulate proteins involved in vesicle fusion

M. tuberculosis is an intracellular pathogen that interferes with phagosome maturation

and fusion with lysosomes. Thus proteins mediating phagosome-endosome or -

lysosome fusion termed SNARE-proteins, represent promising targets for

manipulation and intracellular bacteria have been suggested to interfere with

expression or function of these proteins in order to secure survival inside host-cells.

However, little is known about SNARE proteins in the process of phagocytosis and

phagosome maturation.

As outlined, vesicle fusion is executed by soluble SNARE (N-ethylmaleimide-sensitive

factor attachment protein receptor) proteins. These membrane-anchoring proteins

localize to and function in diverse endomembrane systems in which docking and fusion

between membranes takes place [144]. The SNARE superfamily comprises 36

members in humans with 2 subgroups: The syntaxin and SNAP25 families contain a

conserved glutamine (Q) residue at a central position of the SNARE motif and are

therefore called Qa- and Qbc-SNAREs, respectively. Members of the vesicle-

associated membrane protein (VAMP) family contain a conserved arginine (R) residue

at the same position and are therefore called R-SNAREs [145]. For vesicle fusion,

several Q-SNAREs at target membranes have to assemble and form an acceptor

complex. Acceptor complexes then interact with the vesicular R-SNAREs mediating

vesicle fusion.

Our mass spectrometry results revealed that TDM bead phagosomes accumulate the

Q-SNARE SNAP23 and the R-SNARE VAMP3. Verification of proteomic data by

Western Blot confirmed enrichment of SNARE proteins to TDM bead phagosomes

However, preliminary depletion experiments of VAMP3 by siRNA in macrophages and

subsequent infection with M. tuberculosis suggested that VAMP3 is involved in

phagocytic processes rather than survival of the pathogen.

SNAP23 is a Qbc-SNARE protein located at the plasma membrane and recycling

endomembranes. Together with Syntaxin4, SNAP23 mediates fusion of recycling

endosome membranes bearing VAMP3 with the plasma membrane at the point of

phagocytic-cup formation [146]. However, little is known about its involvement in

membrane fusion with intracellular vesicles during and after phagocytosis. Recently,

Discussion

110

Sakurai et al. could show that overexpression of SNAP23 in macrophages promoted

phago-lysosome fusion indicating a role for SNAP23 in phagosome maturation [104].

SiRNA-mediated reduction of SNAP23 reduced the number of phagocytic events and

delayed phagosome maturation. Furthermore, VAMP7 was identified as cognate R-

SNARE of SNAP23. Taken together, these results revealed that SNAP23 is involved

in phagosome formation and maturation by forming several different SNARE

complexes at the plasma and/or phagosomal membranes.

The R-SNARE VAMP3, also termed cellubrevin, localizes to early and recycling

endosomes and promotes exocytosis of proinflammatory cytokines at the phagocytic

cup when docking to its cognate Q-SNARE, syntaxin-4 and SNAP23 at the plasma

membrane [147]. VAMP3-mediated fusion of vesicles near phagocytic cups has been

revealed to facilitate growth of pseudopods during phagocytosis [148]. However,

BMDM from VAMP3 KO mice revealed that VAMP3 is not essential for phagocytosis

of latex beads but only decelerates phagocytosis through mannose- and β-glucan

receptors [149]. In contrast, our results suggested that cells lacking VAMP3 take up

more M. tuberculosis bacteria as controls whereas survival of M. tuberculosis was not

altered upon VAMP3 depletion. This is in agreement with results from Castañeda-

Ramírez et al. showing that depletion of VAMP3 did not alter survival of Brucella in

J774.A1 macrophages [147]. Furthermore, Fratti et al. demonstrated that phagosomes

of M. tuberculosis and latex-beads acquire VAMP3 over time but only M. tuberculosis

specifically cleaved and thereby inactivated VAMP3 suggesting VAMP3 as target for

Mycobacteria [150].

On the one hand, enrichment of SNAP23 and VAMP3 to TDM bead phagosomes could

simply reflect the early maturation stage of bead phagosomes since both proteins are

present in the plasma membrane from which early phagosomal membranes derive.

On the other hand, inactivation and consequently, retention of SNAP23 and VAMP3

via TDM could prevent mycobacterial vesicles from fusion with early endosomes.

Additional experiments have to be performed in order to confirm these hypotheses.

Discussion

111

4.4 The actin cytoskeleton is involved in M. tuberculosis-

mediated inhibition of phagosome maturation

In the cytoplasm, actin exists in two states, the monomeric (G-actin) and the

filamentous (F-actin). In a process termed actin treadmilling, several G-actin proteins

bind ATP and initiate the nucleation process. At the growing end, termed barbed ends

(+), ATP-bound G-actin monomers nucleate to form filamentous actin. At the pointed

end (-), filamentous actin depolymerises as G-actin monomers hydrolyse ATP and

dissociate.

However, spontaneous nucleation of actin filaments is relatively inefficient. Thus, cells

actively regulate de novo assembly and disassembly of actin filaments as well as their

organisation into functional higher networks with the help of actin-binding-proteins

(ABPs). Generally, ABPs are polypeptides, which in response to signalling cues

undergo conformational changes, and further transmit these signals to downstream

cytoskeletal partners and membranes [151]. Formins and tandem W domain-based

filament nucleators can nucleate F-actin de novo while NPFs-Arp2/3 complexes

mediate branched actin network formation from F-actin mother filaments. Several

NPFs are known that interact with Arp2/3: WASP family verprolin-homologous protein

(WAVE), Wiskott–Aldrich syndrome protein (WASP), neural WASP (NWASP), WASP

and SCAR homologue (WASH), junction-mediating and regulatory protein (JMY) and

cortactin [152]. Regulated by the Rho-family GTPase Rac, WAVE-Arp2/3 complexes

drive branched actin filament formation at lamellipodial protrusions. WASP/NWASP-

Arp2/3 complexes function under the control of Cdc42 for the formation of branched

actin filaments for phagocytosis, endocytosis and exocytosis, adhesion and podosome

formation and trafficking within and from the Golgi apparatus [153]. WASH is a recently

discovered NPF that has been found on endosomes, where it stimulates ARP2/3

activity and controls endosomal sorting [154]. WASH is part of a ~500 kDa complex

(termed WASH complex) with FAM21, SWIP (strumpellin- and WASH-interacting

protein; also known as KIAA1033), strumpellin and CCDC53 (coiled-coil domain-

containing protein 53) and contributes to cargo sorting and scission of transport

intermediates destined for most endosomal routes [152]. Recently, Fam21 was

discovered to link the WASH-complex to endosomes by binding VPS35 of the retromer

complex [155].

Discussion

112

4.4.1 β-actin accumulates on TDM bead and M. tuberculosis phagosomes

Our proteomics data showed that in control and TDM bead phagosomes, actin was

one of the most abundant proteins. Thus, we investigated the localization of actin in

bead infected macrophages and observed that TDM bead phagosomes accumulated

actin rims 30 minutes post infection in RAW264.7 macrophages. In contrast, control

beads were devoid of actin. Quantification revealed that circa 12 % of TDM bead

phagosomes were positive for actin but less than 1 % of control beads.

Isolated latex-bead phagosomes (LPBs) have been described before to initiate actin

assembly in vitro in the presence of ATP and cytosol. Moreover LBPs were revealed

to accumulate actin in vivo [156]–[159]. In the latter studies, actin was localized to latex

beads in a similar pattern to TDM bead phagosomes. In contrast to our findings, actin

accumulation was strongest on late-maturation stage LBPs (1-24 hpi) and suggested

to have a role in fusion of late endosomes to phagosomes and actin-based motility. In

contrast, Lerm et al. suggested that actin depolymerization is important for phagosome

maturation of IgG-opsonized yeast in neutrophils [160]. The discrepancies may be due

to the different experimental systems used. The process of phagocytosis and

phagosome maturation and the underlying cellular processes highly depend on the

phagocytic particle studied. The particles used in these studies were latex beads or

IgG-opsonized particles, which may bind to different receptors than BSA or TDM

beads resulting in different phagocytosis and phagosome biogenesis processes.

In addition to TDM bead, we also revealed that phagosomes of M. tuberculosis

accumulated actin, although in a rather punctate pattern. Several intracellular

pathogens enrich actin to their phagosomes to establish and maintain an intracellular

niche [164], [165]. Internalized Salmonella reside in F- actin-coated phagosomes,

thereby escaping elimination by macrophages [163]. Moreover, the intracellular

parasite Leishmania donovani secures its intracellular survival by forming a coat of F-

actin in a Cdc42- and Rac1-dependent manner [164]. Furthermore, in the

Dictyostelium discoideum model system, phagosomes with M. marinum were entirely

associated with actin whereas in mammalian BV2 macrophages, actin appeared as a

punctual pattern resembling our findings [103]. Therefore, M. marinum recruits WASP

to its phagosomal membrane. In addition, recently the gene product Rv1626 of M.

tuberculosis was shown to interact with Arp2/3 [165]–[167].

Thus, the established default ability of phagosomes to nucleate actin, the well

established strategy of actin nucleation by several pathogens to evade phagosome

maturation and the accumulation of actin on TDM bead and M. tuberculosis

Discussion

113

phagosomes indicates that M. tuberculosis uses the default actin accumulation of

premature phagosomes trough TDM.

4.4.2 Removal of β-actin drives M. tuberculosis into a functional phago-

lysosome

To analyze the function of actin accumulation at M. tuberculosis phagosomes for

mycobacterial survival inside macrophages, actin was removed using Latrunkulin A,

which promoted maturation of M. tuberculosis phagosomes. At later infection stages,

LatA treatment had no effect, suggesting that the mycobacterial compartment

undergoes a phase, in which accumulation of F-actin is essential for phagosomal

arrest. This is in contrast to other studies showing that only mature phagosomes and

late endosomes nucleate actin in order to facilitate fusion with lysosomes [158], [159].

However our results are in accordance with studies reporting nucleation of actin by

immature phagosomes [157], [168]. Liebl & Griffiths concluded that actin accumulation

on immature phagosomes forms a fusion barrier to block fusion with vesicles

promoting maturation whereas Guérin et al. suggested that actin is required for fusion

with early endosomes. Our results confirm the hypothesis of Liebl & Griffith since

removal of actin with Latrunkulin A resulted in phagosome maturation and concurrent

reduced survival of M. tuberculosis macrophages.

We therefore conclude that F-actin nucleation on the mycobacterial phagosome is

distinctly regulated over the course of infection: Actin polymerization at early time

points is required for blocking fusion with endosomes but is suppressed at later stages

to block fusion of the established niche, competent for mycobacterial replication, with

lysosomes. This is an evolutionary conserved mechanism used by M. tuberculosis and

M. marinum in both mammalian macrophages and the social amoeba Dictyostelium

discoideum [103].

4.4.3 Proteins regulating actin treadmilling are important for survival of M.

tuberculosis in macrophages but not the WASH-complex

Phagosomal membranes carry all the machinery sufficient to assemble F-actin de

novo by the activity of actin-binding peripheral membrane proteins [140], [169]. Up to

date, several actin-binding-proteins were identified on phagosomes: annexins, talin,

vinculin, α-actinin, myosins, MARKS (myristoylated, alanine-rich C-kinase substrate),

profiling, TACO/coronin1 and Arp2/3 [169]. However, most of these proteins were only

described to be present on isolated LBPs and not further analyzed for their role in actin

assembly during phagosome maturation. As an exception, ezrin/moesin was shown to

Discussion

114

mediate de novo actin assembly on LPBs [156]. Furthermore, TACO/coronin1 was

dispensable for F-actin dependent processes, but essential for the inhibition of M.

tuberculosis and M. bovis BCG phagosome maturation [170], [171]. All mentioned

ABPs except for vinculin and MARKS were identified in our bead phagosomes with

profilin1, cofilin1 and annexins A1 and A6 up-regulated in TDM bead phagosomes.

Our mass spectrometry analysis showed that profilin1 and cofilin1 were threefold

accumulated on TDM bead phagosomes compared to controls as corroborated by

Western blot.

In a process called actin treadmilling, G-actin binds ATP and assembles to F-actin.

ATP hydrolysis of G-actin leads to de-polymerization of the existing filament at the

pointed end. Actin treadmilling is assisted by cytoplasmic G-actin binding proteins

cofilin and profilin. Profilin interacts with actin nucleators and accelerates ADP to ATP

exchange to promote incorporation of ATP-bound G-actin to an existing microfilament.

Cofilin accelerates dissociation of ADP-bound G-actin from pointed ends. Thus the

main function of both proteins is to regulate the dynamics of actin inside the cytoplasm

by promoting actin polymerization and depolymerisation, respectively. Although their

function in phagocytosis is established, their role in actin polymerization for

endosomal-phagosomal trafficking is only beginning to become clearer. Experiments

revealed that cofilin together with the WASP-Arp2/3-complex, has a role in

phagocytosis of Candida albicans by neutrophils [172]. Similarly, profilin is required for

Fc-receptor mediated phagocytosis [173]. Recently, cofilin was shown to participate in

actin depolymerisation hindering fusion of Legionella phagosomes with lysosomes in

a caspase regulated manner [174]. In addition, profilin and cofilin were demonstrated

to be essential for actin coat formation at LBPs in the D. discoideum model system

[175]. Bielig et al. lately suggested a link between bacterial induced changes in actin

dynamics and the intracellular receptor NOD1 via the cofilin phosphatase SSH1 [176].

Accumulation of profilin1 and cofilin1 indicates that actin is actively assembled and

disassembled on TDM bead phagosomes. The fact that both proteins are enriched on

TDM bead phagosomes further suggests that levels of actin are maintained. This is in

accordance with the presence of actin found on phagosomes of TDM bead and M.

tuberculosis. Depolymerisation of actin with LatrunkulinA and knock-down of profilin1

and cofilin1 resulted in debased survival of M. tuberculosis in macrophages. This

suggests that F-actin assembly is important for M. tuberculosis to establish and

maintain a proliferative niche in macrophages. However, knock-down of WASH1 did

not inhibit survival of M. tuberculosis. This suggests that actin polymerization at the

Discussion

115

mycobacterial phagosome uses other actin-nucleators to establish the protective actin-

coat. These results are in contrast with previous finings in M. marinum infected D.

discoideum amoeba [103]. In wash-/- amoeba, vacuoles of M. marinum were

significantly more associated with the v-H+-ATPase and thus more mature than in WT

amoeba [103]. These conflicting results may be due to the fact that knock-down of

WASH1 did not completely remove WASH1 from RAW264.7 macrophages.

Remaining WASH1 might have been sufficient to establish the protective actin-coat.

Moreover, the WASH-complex might be active even in the absence of WASH1. Since

in addition to WASH, other ABPs such as annexinA1 and annexinA6 were found at

TDM bead phagosomes, these actin-nucleators might be sufficient to establish actin

coats at M. tuberculosis phagosomes.

Discussion

116

4.5 TDM-mediated inhibition of phagosome maturation changes

the lipid composition of bead phagosomes

Phospholipids, glycolipids and cholesterol are membrane lipids that constitute the

plasma membrane, compartmentalize cells and coordinate signaling, targeting and

trafficking events in the course of phagosome generation and maturation. Certain lipid-

species are required both, as substrates for and as activators of enzymes responsible

for the generation of important second messengers [177]. Lastly, specific lipid head-

groups recruit proteins possessing lipid-binding domains and/or confer curvature and

charge to the membrane surface, promoting electrostatic attraction and retention of

proteins [177]. However, lipids have long been neglected players in phagosome

biology. Thus their role in phagocytosis and phagosome maturation is only beginning

to be understood. So far, the best studied and probably most important function of

lipids is coordination of recruitment and retention of key proteins for phagocytosis.

Shortly after nascent phagosome formation, the phagocytic membrane resembles the

plasma membrane. But maturation entails further changes in the lipid composition of

the phagosomal membrane, raising the possibility of additional fluctuations in surface

charge and consequently, protein targeting [177].

In the present work, the lipid composition of control bead phagosomes was compared

to TDM bead phagosomes in order to reveal TDM-mediated changes in the bead

phagosome lipidome regarding twelve different lipid classes. To our knowledge this is

the first analysis of the lipid composition of bead phagosomes that covered more than

six different lipid classes.

In 1994, Desjardins and co-workers dissected the lipid-composition of early (2 hpi) and

late (24 hpi) LBPs [178]. They described SM, PC, PS, PI, PE and LBPA. Early LBPs

contained less SM and PE and more PC than late LPBs. These differences were not

confirmed by our results using control and TDM beads. The amounts of SM, PE and

PC were similar in control and TDM bead phagosomes. In contrast, to the work of

Desjardins et al. showing unchanged PS and LBPA levels, our analyses revealed that

PS was accumulated in TDM whereas LBPA was reduced compared to control bead

phagosomes. In addition cholesterol, which was not analyzed in the former study, was

accumulated in TDM bead phagosomes. Analysis on the lipid-species level confirmed

these results but further revealed up-regulation of certain ceramide and

sphingomyeline species in TDM bead phagosomes compared to controls.

Discussion

117

Based on our proteomic analysis of control and TDM bead phagosomes and previous

studies using the same model, we assume that, due to TDM inhibitory effects, TDM

are less mature than control bead phagosomes [91]. However, it is questionable

whether both experiments can be compared since Desjardins et al. determined 2 hpi

and 24 hpi as early or late time points, respectively. The time points analyzed in our

study are significantly different. In addition, Desjardins et al. used latex-beads which

may use different phagocytic receptors than BSA and TDM beads.

Of the three lipid classes accumulated in TDM bead phagosomes, cholesterol is best

described for its role in phagosome maturation. Together with shpingolipids and

several proteins, cholesterol forms so called lipid microdomains which are implicated

in signal transduction and membrane trafficking. Yet, the distribution of cholesterol in

maturing endosomes is not clear. The plasma membrane contains relatively high

amounts of cholesterol and glycosphingolipids [179]. Thus accumulation of cholesterol

in TDM bead phagosomes compared to controls may simply reflect their early

phagosomal stage maintaining plasma membrane-like composition. Other studies

suggested that lysosomes harbor little cholesterol while late endosomes are

cholesterol rich [177]. This hypothesis could be confirmed by our results as TDM bead

phagosomes harbored more cholesterol than control bead which are enriched for late

endosomal/lysosomal marker proteins. Further, accumulation of cholesterol was

shown to inhibit phago-lysosomal fusion but not fusion of early endosomes with

phagosomes [180]. The authors suggested that accumulation of cholesterol rendered

phagosomal membrane organization non-fusogenic by inactivation of important

fusion-mediating proteins such as Rab7. Thus accumulation of cholesterol to

phagosomes is favourable for pathogens to reside in early phagosomes in order to

survive within phagocytes. Our results confirm this hypothesis as TDM bead

phagosomes accumulating cholesterol are delayed in phago-lysosomal fusion.

The role of cholesterol in mycobacterial infection has been studied before: Cholesterol

is indispensable for uptake of Mycobacteria into phagocytes. Furthermore, cholesterol

is essential for maintaining the phagosome membrane closely apposed to the

mycobacterial surface and therefore for prevention of phagosome maturation [181]. In

this study, removal of cholesterol promoted phago-lysosome fusion and also

diminished lipid-domain containing sphingomyelins and binding of lipid-microdomain

interacting proteins. Cholesterol can also be used as carbon source as M. tuberculosis

was shown to harbour genes required for cholesterol intake and catabolism [182].

Discussion

118

Therefore, establishing a niche with access to cholesterol can promote intracellular

growth.

The fact that TDM bead phagosomes accumulated cholesterol indicates that TDM is

involved in reorganization of phagosomal membranes for the inhibition of phagosome

maturation. TDM may accumulate cholesterol because (i) it ensures a tight apposition

important for the pathogen to deliver virulence factors across the phagosomal

membrane (ii) cholesterol can attract cytosolic proteins as annexins that mediate

accumulation of actin to establish an actin coat inhibiting phagosome maturation, (iii)

cholesterol serves an important nutrient source or (iv) high levels of cholesterol change

the phagosomal membrane organization in order to avoid activation or recruitment of

phagosome maturation effector proteins.

About 15 % of total plasma membrane lipids in the inner leaflet are constituted by PS

species. In stark contrast, little PS is detectable in the outer leaflet of healthy cells

plasma membranes [177]. This asymmetric distribution is lost during apoptosis, and

flipping of PS to the outer surface serves to identify cells destined for efferocytosis and

degradation [177]. However, little is known about PS distribution, metabolism, and

function during the course of phagosome maturation [177]. The study of Desjardins

and co-workers suggested that amounts of PS remain relatively unchanged during in

the course of phagosome maturation [178]. In contrast, our results show that PS was

accumulated to TDM bead phagosomes compared to controls [183]. In studies by

Yeung et al., the amount of PS on sheep red-blood-cell phagosomes was suggested

to be maintained through fusion with endosomes and lysosomes. PS in early

phagosomes most probably derives from the plasma membrane. But the observation

that PS was still present after 1 hour post infection, indicated that PS is either

maintained in the phagosomal membrane or delivered to the phagosome during

maturation through fusion with endosomes from the recycling or late endocytic

pathway [183]. Consequently, phagosomes of intracellular pathogens as Legionella

and Chlamydia, interfering with phagosome maturation, were shown to be devoid of

PS [183]. However this was not the case for TDM bead phagosomes indicating that

these phagosomes might have access to the endocytic pathway further corroborated

by late endocytic marker proteins in the TDM bead phagosome proteome. Indeed

mycobacterial phagosomes have, although arrested at an early phagosomal state,

access to the endocytic pathway and an intense exchange with the plasma membrane

[115],[184]. Moreover, since PS is an anionic lipid, Yeung et al. also suggested that

PS serves to recruit cationic proteins such as Src kinases to endosomal/phagosomal

Discussion

119

membranes. Taken together, retention of PS may recruit or retain proteins involved in

controlling phagosome biogenesis.

Lysobisphosphatic acid (LBPA) is an unusual lipid found in high amounts in late

endosomes and intra-luminal-vesicles (ILVs) of multivesicular bodies but is not

detected elsewhere in the cell [185]. Though, it is frequently used as marker for

endosome/phagosome maturation. In late endosomes, LBPA makes up 15 mol % of

all total cellular phospholipids [179]. In control bead phagosomes we found 22 mol %

but only 14 mol % in TDM bead phagosomes. This indicates that TDM bead

phagosomes had less access to late endosomes than controls.

The main LBPA isoform detected in several cell lines was shown to be 2,2’-dioleoyl

LBPA [185]–[189]. Thus it may also be the major biologically active species. This was

confirmed by our results as LBPA 38:2 was revealed to be the most abundant lipid-

species found in bead phagosomes. LBPA has two identified protein interaction

partners: ALIX (also termed programmed cell death interacting protein 6, PDCD6IP)

and Hsp70 [190]. ALIX is recruited to endosomes via interactions with LBPA

suggesting that apart from ILVs, LBPA can be present on the cytoplasmic leaflet of

late endosomes/phagosomes. Our proteomic analysis identified 4 EUSC for PDCD6IP

and Hsp70 in TDM bead phagosomes and 2 EUSC of Hsp70 in control samples.

Interaction of 2,2’-dioleoyl LBPA with ALIX triggers ILVs formation within acidic

liposomes. However, the most important function of LBPA-ALIX interaction is

cholesterol homeostasis [191]. LBPA-ALIX interaction mediates the back-fusion of

cholesterol-rich ILV in late endosomes ensuring cholesterol homeostasis. In

conclusion, exclusion of LBPA may maintain cholesterol content of mycobacterial

phagosomes.

Discussion

120

4.6 Conclusion: TDM-mediated changes of the phagosomal

proteome and lipidome – potential interaction partners or

innocent bystanders?

Overall 1054 proteins and 12 different lipid classes with 312 different lipid species were

detected in control and TDM bead phagosomes. All proteins and lipids detected raise

the question whether their presence is a consequence of TDM-mediated manipulation

of phagosome maturation or if they are maturation stage specific and thus innocent

bystanders. However, the fact that certain proteins associate with the actin

cytoskeleton suggest a role for the actin cytoskeleton in TDM-mediated inhibition of

phagosome maturation.

At this point, one should consider distribution of TDM on beads. We assumed a rather

uniform distribution of TDM on BSA beads. Yet, experiments in artificial bilayers

suggested that TDM is organized in domains with unusual high compared to control

bilayers (Thomas Gutsmann, personal communication). Further one must account the

orientation of TDM regardless uniform or domain-like distribution on BSA beads. We

supposed that TDM binds to hydrophobic pockets of BSA via its mycolic acids so that

the trehalose moiety stacked out. Moreover, previous experiments revealed that TDM

is firmly attached to BSA beads [91]. Binding with both mycolic acids would allow the

trehalose-headgroup to interact with host-cell derived interaction partners (1, Figure

4.1). Nevertheless, binding with one mycolic acid but also detachment of TDM from

BSA cannot be totally excluded (2, 3 Figure 4.1). This would allow TDM to intercalate

to the phagosomal membrane.

We hypothesize that TDM may be responsible for the accumulation of actin around M.

tuberculosis phagosomes to establish inhibition of phagosome maturation. Regardless

orientation of TDM, phagosomal membrane lipids as cholesterol and PS might be

direct interaction partners of TDM. Retention of certain lipids may attract indirect

interaction partners as cytosolic proteins involved in actin accumulation (such as

annexins or cofilin1) to establish the protective actin coat. When intercalating into the

phagosomal membrane, TDM might also directly manipulate cytosolic actin-binding-

proteins and/or nucleation-promoting-factors. TDM inside phagosomal membranes

may also inactivate SNARE proteins responsible for membrane fusion events.

Moreover, retention of cholesterol itself might contribute to inhibition of phagosome

Discussion

121

maturation as accumulation of the lipid was shown to permit membrane fusion

processes [180].

Figure 4.1: Scheme of the potential function of TDM-mediated inhibition of phagosome maturation. TDM may (1) interact with host-cell proteins or lipids via its trehalose headgroup, (2) by intercalation of 1 mycolic acid with the phagosomal membrane or (3) by intercalation of the whole molecule to the phagosomal membrane. TDM may exert several mechanisms to inhibit phagosome maturation. TDM could influence the phagosomal membrane and retain phosphatidylserine (PS) and cholesterol (chol). PS and cholesterol are major binding partners for annexinA1 and annexinA6. Annexins then may induce actin nucleation at the phagosomal membrane by recruiting actin-binding-proteins and/or nucleation-promoting-factors as profilin1 (pfl1) to establish actin rims. Actin rims might inhibit fusion of the phagosome with multivesicular bodies (MVBs) and lysosomes. Furthermore TDM might retain cholesterol on the phagosomal membrane to inhibit fusion with MVBs and lysosomes because the lipid itself was shown to inhibit fusion processes. SNAREs SNAP23 and VAMP3 indicate the fusogenic properties of TDM bead phagosomes with early and intermediate endosomes but might also be manipulated and thus inactivated by TDM intercalating to the phagosomal membrane.

Discussion

122

4.7 Perspectives

In this PhD-thesis, a protocol for isolation and purification of magnetic lipid-coated

bead phagosomes was developed. Still, the protocol has its drawbacks. A critical step

is the trypsin digestion because it may remove potential TDM interaction partners from

the outer surface of the phagosomal membrane. Emphasis should be put on

development of another way to detach bead phagosomes from each other. In other

studies, bead phagosomes were freed by incubation with ATP that leads to

depolymerization of actin networks possibly gluing bead-phagosomes together.

Another critical step is the label-free approach for quantification of proteins. For the

future, macrophages should be metabolically labeled by stable-isotope-labeling-in-

cell-culture (SILAC) in order to establish a fully-quantitative method. Furthermore,

purification by FACS sorting should include IF-staining of bead phagosomes for

LAMP1 in order to sort bead phagosomes according to their maturation state in

addition to size.

Herein 34 proteins were discovered to be accumulated to TDM bead phagosomes

compared to controls. Of the latter, 6 were chosen for further analysis. An ongoing

attempt should be the analysis of remaining 27 proteins for their potential function in

TDM-mediated inhibition of phagosome maturation using IF-staining, Western blot and

siRNA knock-down/CFU experiments. Moreover, the siRNA knock-down/CFU

experiments shown herein were performed once. To further verify the results, the

experiment should be repeated at least three times, also for statistical reasons.

We could show actin accumulation on TDM bead and M. tuberculosis phagosomes

has a role in inhibition of phagosome maturation. SiRNA/CFU experiments suggested

that profilin1 and cofilin1 are important for survival of M. tuberculosis whereas WASH1,

annexinA6, SNAP23 and VAMP3 were not. Ongoing experiments must elucidate the

NPF of actin on phagosomes of TDM beads and M. tuberculosis. Potential candidates

are annexins as they are well known actin nucleators. Apart from analysing the survival

of M. tuberculosis in absence of candidate proteins, one must also check whether TDM

and the pathogen are still able to nucleate actin on their phagosomes and inhibit

phagosome maturation. This could be accomplished by siRNA knock-down and

subsequent IF-staining of phagosomes of TDM beads and M. tuberculosis by

lysotracker, LAMP1 and actin. The latter experiment is most important as it would

Discussion

123

directly show the link between the single virulence factor TDM and its interaction

partner.

Further experiments to evaluate the role of lipids in TDM-mediated inhibition of

phagosome maturation are difficult. In contrast to most proteins, depletion of lipids has

extensive impact on cell physiology. In addition, specific antibodies to certain lipids are

rare. However, ongoing experiments could include staining of TDM bead phagosome

membranes in macrophages using fluorescent dyes specific for cholesterol rich

domains. Moreover, for comparison with TDM bead phagosome samples, the lipid

composition of M. tuberculosis phagosomes should be analyzed.

To conclude, this thesis suggests that TDM inhibits phagosome maturation by

manipulating the cells actin cytoskeleton. However, experiments are required to further

corroborate this hypothesis. Moreover, this thesis provides several other interesting

TDM targets worth analyzing.

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145

149. Interferon-induced transmembrane protein 3

sp|Q9CQW9|IFM3_MOUSE 6 18

150. Ubiquitin-associated protein 2-like sp|Q80X50|UBP2L_MOUSE 2 6

151. Ribosomal RNA-processing protein 1 homolog A

tr|E9QL52|E9QL52_MOUSE 2 6

152. Integrin alpha-4 tr|E9QK91|E9QK91_MOUSE (+1)

3 9

153. Ras-related protein Rap-1b sp|Q99JI6|RAP1B_MOUSE 3 9

154. Transmembrane emp24 domain-containing protein 9

sp|Q99KF1|TMED9_MOUSE 2 6

155. High affinity immunoglobulin epsilon receptor subunit gamma

sp|P20491|FCERG_MOUSE 2 6

156. Mitochondrial import receptor subunit TOM34

sp|Q9CYG7|TOM34_MOUSE 3 9

157. Isoform 2 of Regulation of nuclear pre-mRNA domain-containing protein 2

sp|Q6NXI6-2|RPRD2_MOUSE 2 6

158. Gamma-aminobutyric acid receptor-associated protein-like 2

sp|P60521|GBRL2_MOUSE 2 6

159. Peptidyl-prolyl cis-trans isomerase FKBP11

sp|Q9D1M7|FKB11_MOUSE 2 6

160. Apoptosis regulator BAX sp|Q07813|BAX_MOUSE 2 6

161. 17beta-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase

tr|Q99N15|Q99N15_MOUSE 3 8

162. Tyrosine-protein kinase BAZ1B sp|Q9Z277|BAZ1B_MOUSE 3 8

163. Protein Ythdf2 tr|E9Q2W5|E9Q2W5_MOUSE 3 8


165. Protein Dnajc13 tr|D4AFX7|D4AFX7_MOUSE 2 5


sp|P63024|VAMP3_MOUSE 8 20

167. FK506-binding protein 15 sp|Q6P9Q6|FKB15_MOUSE 2 5

168. Periplakin sp|Q9R269|PEPL_MOUSE 2 5

169. Copine VIII tr|B2RS65|B2RS65_MOUSE 2 5

170. Ubiquitin-like modifier-activating enzyme 1

sp|Q02053|UBA1_MOUSE 2 5

171. Hematopoietic lineage cell-specific protein

sp|P49710|HCLS1_MOUSE 2 5

172. Synaptosomal-associated protein tr|Q9D3L3|Q9D3L3_MOUSE 7 17

173. Cytochrome c oxidase subunit 4 isoform 1, mitochondrial

sp|P19783|COX41_MOUSE 5 12

174. Protein ERGIC-53 sp|Q9D0F3|LMAN1_MOUSE 5 12

175. Voltage-dependent anion-selective channel protein 3

sp|Q60931|VDAC3_MOUSE 5 12

176. MKIAA1699 protein (Fragment) tr|Q69ZD1|Q69ZD1_MOUSE 5 12

177. Signal recognition particle 68 kDa protein

sp|Q8BMA6|SRP68_MOUSE 8 19

178. Cation-dependent mannose-6-phosphate receptor

sp|P24668|MPRD_MOUSE 3 7

179. Vacuolar protein sorting-associated protein 11 homolog

sp|Q91W86|VPS11_MOUSE 3 7

180. Pescadillo homolog tr|Q5SQ20|Q5SQ20_MOUSE 6 14

181. Elongation factor 2 sp|P58252|EF2_MOUSE 4 9

182. Neutrophil cytosol factor 2 sp|O70145|NCF2_MOUSE 4 9

183. Multifunctional protein ADE2 sp|Q9DCL9|PUR6_MOUSE 4 9


147

224. Tumor susceptibility gene 101 protein sp|Q61187|TS101_MOUSE 10 18

225. Adenylyl cyclase-associated protein 1 sp|P40124|CAP1_MOUSE 5 9

226. Peroxisomal membrane protein 11B tr|D6RFQ2|D6RFQ2_MOUSE 5 9

227. Peroxiredoxin 1 (Fragment) tr|B1AXW5|B1AXW5_MOUSE 14 25

228. ADP-ribosylation factor-like protein 8A

sp|Q8VEH3|ARL8A_MOUSE 4 7

229. ADP-ribosylation factor 4 sp|P61750|ARF4_MOUSE (+1) 12 21

230. contamination K1H1_HUMAN 8 14

231. Eukaryotic initiation factor 4A-III sp|Q91VC3|IF4A3_MOUSE 8 14

232. Beta-parvin sp|Q9ES46|PARVB_MOUSE 4 7

233. Ras-related protein Rab-7a sp|P51150|RAB7A_MOUSE 35 61

234. Eukaryotic translation initiation factor 3 subunit E

sp|P60229|EIF3E_MOUSE 3 5

235. Ras-related protein Rab-2A sp|P53994|RAB2A_MOUSE 12 20

236. Inositol 1,4,5-trisphosphate receptor type 3

sp|P70227|ITPR3_MOUSE 6 10

237. Guanine nucleotide-binding protein G(k) subunit alpha

sp|Q9DC51|GNAI3_MOUSE 6 10

238. Small subunit processome component 20 homolog

tr|E9QK83|E9QK83_MOUSE 6 10


sp|O70480|VAMP4_MOUSE 6 10

240. Probable cation-transporting ATPase 13A2

sp|Q9CTG6|AT132_MOUSE 6 10

241. Actin-related protein 3 sp|Q99JY9|ARP3_MOUSE 3 5

242. Stromal cell-derived factor 2-like protein 1

sp|Q9ESP1|SDF2L_MOUSE 3 5

243. Metaxin-2 sp|O88441|MTX2_MOUSE 3 5

244. 40S ribosomal protein S18 sp|P62270|RS18_MOUSE (+1) 14 23

245. Actin-related protein 2 sp|P61161|ARP2_MOUSE 11 18

246. Arf-GAP domain and FG repeat-containing protein 2

sp|Q80WC7|AGFG2_MOUSE (+1)

24 39

247. 3-ketoacyl-CoA thiolase A, peroxisomal

sp|Q921H8|THIKA_MOUSE 8 13

248. Probable phospholipid-transporting ATPase IIB

sp|P98195|ATP9B_MOUSE 8 13

249. Translocon-associated protein subunit delta

sp|Q62186|SSRD_MOUSE (+1)

13 21

250. Tripeptidyl-peptidase 1 sp|O89023|TPP1_MOUSE 5 8

251. Fermitin family homolog 3 sp|Q8K1B8|URP2_MOUSE 20 32

252. Protein Rab1 tr|H7BX41|H7BX41_MOUSE 5 8

253. Vesicle-trafficking protein SEC22b sp|O08547|SC22B_MOUSE 5 8

254. Uncharacterized protein tr|E9Q390|E9Q390_MOUSE 19 30

255. Succinyl-CoA ligase [GDP-forming] subunit beta, mitochondrial

sp|Q9Z2I8|SUCB2_MOUSE 7 11

256. Monocyte differentiation antigen CD14

sp|P10810|CD14_MOUSE 16 25

257. Voltage-dependent anion-selective channel protein 1

sp|Q60932|VDAC1_MOUSE 27 42

258. Nicalin tr|D3YU17|D3YU17_MOUSE 9 14

259. Aldehyde dehydrogenase family 3 member B1

sp|Q80VQ0|AL3B1_MOUSE 11 17

260. Ras-related protein Rap-1A sp|P62835|RAP1A_MOUSE 15 23

261. Annexin A7 sp|Q07076|ANXA7_MOUSE (+1)

27 41


152

420. Ras-related GTP-binding protein C sp|Q99K70|RRAGC_MOUSE 12 13

421. Galactocerebrosidase sp|P54818|GALC_MOUSE 13 14

422. Sorting nexin 1 tr|Q6NZD2|Q6NZD2_MOUSE 15 16

423. Elongation factor Tu, mitochondrial sp|Q8BFR5|EFTU_MOUSE 16 17

424. Lactadherin sp|P21956|MFGM_MOUSE (+1)

16 17

425. Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1

sp|Q91YQ5|RPN1_MOUSE 33 35

426. V-type proton ATPase subunit B, brain isoform

sp|P62814|VATB2_MOUSE 85 90

427. Tubulin beta-5 chain sp|P99024|TBB5_MOUSE 68 72

428. 6-phosphofructokinase tr|Q8C605|Q8C605_MOUSE 34 36

429. Elongation factor 1-alpha 1 sp|P10126|EF1A1_MOUSE 35 37

430. Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A

sp|P46978|STT3A_MOUSE 18 19

431. contamination ENO1_YEAST 55 58

432. Phospholipid hydroperoxide glutathione peroxidase, mitochondrial

sp|O70325|GPX41_MOUSE 19 20

433. Sarcoplasmic/endoplasmic reticulum calcium ATPase 2

sp|O55143|AT2A2_MOUSE 39 41

434. Alpha-soluble NSF attachment protein

sp|Q9DB05|SNAA_MOUSE 39 41

435. Aldehyde dehydrogenase, mitochondrial

sp|P47738|ALDH2_MOUSE 20 21

436. Presenilin-1 sp|P49769|PSN1_MOUSE 21 22

437. Alkyldihydroxyacetonephosphate synthase, peroxisomal

tr|A2AL50|A2AL50_MOUSE 22 23

438. EH domain-containing protein 4 sp|Q9EQP2|EHD4_MOUSE 54 56

439. Flotillin-1 sp|O08917|FLOT1_MOUSE 29 30

440. Protein disulfide-isomerase sp|P09103|PDIA1_MOUSE 64 66

441. Tetratricopeptide repeat protein 39B sp|Q8BYY4|TT39B_MOUSE 39 40

442. EH domain-containing protein 1 tr|F6YBI4|F6YBI4_MOUSE 47 48

443. Protein disulfide-isomerase A6 sp|Q922R8|PDIA6_MOUSE 60 61

444. ATP synthase subunit alpha, mitochondrial

sp|Q03265|ATPA_MOUSE 63 64

445. V-type proton ATPase subunit E 1 sp|P50518|VATE1_MOUSE 64 65

446. V-type proton ATPase catalytic subunit A

sp|P50516|VATA_MOUSE 163 165

447. V-type proton ATPase subunit H sp|Q8BVE3|VATH_MOUSE 53 53

448. Protein disulfide-isomerase A3 sp|P27773|PDIA3_MOUSE 52 52

449. Protein Ahnak tr|E9Q616|E9Q616_MOUSE 15 15

450. Prolow-density lipoprotein receptor-related protein 1

sp|Q91ZX7|LRP1_MOUSE 6 6

451. Hexokinase-2 tr|E9Q5B5|E9Q5B5_MOUSE 22 22

452. Histone H2B type 1-F/J/L sp|P10853|H2B1F_MOUSE 3 3

453. Uncharacterized protein tr|E9PZD8|E9PZD8_MOUSE 2 2

454. Keratin, type I cytoskeletal 13 sp|P08730|K1C13_MOUSE 13 13

455. Isoform 2 of Phospholipase D4 sp|Q8BG07-2|PLD4_MOUSE (+1)

18 18

456. Ras-related protein Rab-5C sp|P35278|RAB5C_MOUSE 25 25

457. Vesicle transport through interaction with t-SNAREs homolog 1B

tr|E9Q5B1|E9Q5B1_MOUSE 28 28

458. ATP-binding cassette sub-family A member 3

sp|Q8R420|ABCA3_MOUSE 10 10


159

686. Isoform 2 of Prolyl 4-hydroxylase subunit alpha-1

sp|Q60715-2|P4HA1_MOUSE 11 8

687. Long-chain-fatty-acid--CoA ligase 4 sp|Q9QUJ7|ACSL4_MOUSE 14 10

688. Histone acetyltransferase type B catalytic subunit

sp|Q8BY71|HAT1_MOUSE 14 10

689. Neutrophil cytosol factor 4 sp|P97369|NCF4_MOUSE 21 15

690. Ceroid-lipofuscinosis neuronal protein 5 homolog

sp|Q3UMW8|CLN5_MOUSE 7 5

691. Ubiquitin-associated protein 2 sp|Q91VX2|UBAP2_MOUSE 7 5

692. Desmoglein-1-alpha sp|Q61495|DSG1A_MOUSE 30 21

693. RNA-binding protein 12 sp|Q8R4X3|RBM12_MOUSE 10 7

694. Pro-cathepsin H sp|P49935|CATH_MOUSE 10 7

695. Far upstream element-binding protein 2

sp|Q3U0V1|FUBP2_MOUSE 10 7

696. Minor histocompatibility antigen H13 sp|Q9D8V0|HM13_MOUSE 13 9

697. Keratin, type I cytoskeletal 42 sp|Q6IFX2|K1C42_MOUSE 31 21

698. Niemann Pick type tr|G3X8W9|G3X8W9_MOUSE 18 12

699. Isoform Cytoplasmic of Fumarate hydratase, mitochondrial

sp|P97807-2|FUMH_MOUSE (+1)

12 8

700. Integrin alpha M, isoform CRA_a tr|G5E8F1|G5E8F1_MOUSE 9 6

701. Toll-like receptor 13 sp|Q6R5N8|TLR13_MOUSE 9 6

702. T-complex protein 1 subunit tr|Q3U0I3|Q3U0I3_MOUSE 6 4

703. Lon protease homolog tr|E9Q120|E9Q120_MOUSE 15 10

704. Transcriptional regulator ATRX sp|Q61687|ATRX_MOUSE 3 2

705. Monocarboxylate transporter 1 sp|P53986|MOT1_MOUSE 6 4

706. Nuclear pore complex protein Nup160

sp|Q9Z0W3|NU160_MOUSE 12 8

707. Isocitrate dehydrogenase [NADP] tr|F8WIY0|F8WIY0_MOUSE (+1)

9 6

708. Charged multivesicular body protein 4b

sp|Q9D8B3|CHM4B_MOUSE 9 6

709. Pleckstrin sp|Q9JHK5|PLEK_MOUSE 3 2

710. Unconventional myosin-Va tr|D3Z4J3|D3Z4J3_MOUSE 3 2

711. Zinc finger SWIM domain-containing protein KIAA0913

sp|Q3UHH1|K0913_MOUSE 3 2

712. ADP-ribosylation factor guanine nucleotide-exchange factor 2 (Brefeldin A-inhibited)

tr|A2A5R2|A2A5R2_MOUSE 3 2

713. Stromal membrane-associated protein 2

sp|Q7TN29|SMAP2_MOUSE 6 4

714. Histone H1.3 sp|P43277|H13_MOUSE 3 2

715. Condensin complex subunit 1 sp|Q8K2Z4|CND1_MOUSE 3 2

716. Staphylococcal nuclease domain-containing protein 1

sp|Q78PY7|SND1_MOUSE 3 2


sp|Q9D1C8|VPS28_MOUSE 9 6

718. DNA replication licensing factor MCM2

sp|P97310|MCM2_MOUSE 3 2

719. Cytosol aminopeptidase sp|Q9CPY7|AMPL_MOUSE 3 2

720. Protein 9030624J02Rik (Fragment) tr|D3YW19|D3YW19_MOUSE 6 4

721. Copine-2 sp|P59108|CPNE2_MOUSE 3 2

722. Gelsolin sp|P13020|GELS_MOUSE (+1) 3 2

723. Isoform 2 of Telomere-associated protein RIF1

sp|Q6PR54-2|RIF1_MOUSE (+1)

3 2

724. Nucleobindin-1 sp|Q02819|NUCB1_MOUSE 3 2


165

921. Protein Nup98 tr|E9Q4S2|E9Q4S2_MOUSE 3 0

922. Dedicator of cytokinesis protein 5 sp|B2RY04|DOCK5_MOUSE 2 0

923. FH1/FH2 domain-containing protein 1 sp|Q6P9Q4|FHOD1_MOUSE 2 0


sp|Q91WD5|NDUS2_MOUSE 2 0

925. DNA mismatch repair protein Msh2 sp|P43247|MSH2_MOUSE 2 0

926. Glycerol-3-phosphate dehydrogenase, mitochondrial

sp|Q64521|GPDM_MOUSE 2 0

927. CTP synthase 1 sp|P70698|PYRG1_MOUSE 2 0

928. ADP-ribosylation factor-like protein 1 sp|P61211|ARL1_MOUSE 2 0

929. Exportin-5 sp|Q924C1|XPO5_MOUSE 2 0

930. Neighbor of COX4 sp|O70378|CX4NB_MOUSE 2 0

931. Nuclear pore complex protein Nup107

tr|E9Q4V9|E9Q4V9_MOUSE 3 0

932. Probable ATP-dependent RNA helicase DHX58

sp|Q99J87|DHX58_MOUSE 2 0

933. Uncharacterized protein tr|E9QK78|E9QK78_MOUSE 2 0

934. Isoform 2 of RNA-binding protein 39 sp|Q8VH51-2|RBM39_MOUSE (+1)

3 0

935. Sorting and assembly machinery component 50 homolog

sp|Q8BGH2|SAM50_MOUSE 2 0

936. Serpin B5 sp|P70124|SPB5_MOUSE 4 0

937. Arginine-glutamic acid dipeptide repeats protein

sp|Q80TZ9|RERE_MOUSE 2 0

938. Nucleolar GTP-binding protein 1 sp|Q99ME9|NOG1_MOUSE 2 0

939. Protein transport protein Sec23B sp|Q9D662|SC23B_MOUSE 7 0

940. Dihydrolipoyl dehydrogenase, mitochondrial

sp|O08749|DLDH_MOUSE 2 0

941. Cytochrome sp|P56395|CYB5_MOUSE (+1) 2 0

942. RNA 3'-terminal phosphate cyclase sp|Q9D7H3|RTC1_MOUSE 2 0

943. Replication factor C subunit 2 sp|Q9WUK4|RFC2_MOUSE 7 0

944. Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial

sp|Q9D6R2|IDH3A_MOUSE 2 0

945. Ran-binding protein 3-like sp|Q6PDH4|RNB3L_MOUSE 2 0

946. Structural maintenance of chromosomes protein 3

tr|E9Q762|E9Q762_MOUSE 3 0

947. Rho guanine nucleotide exchange factor 10-like protein

sp|A2AWP8|ARGAL_MOUSE 2 0

948. Eukaryotic translation initiation factor 3 subunit D

sp|O70194|EIF3D_MOUSE 2 0

949. Phosphoenolpyruvate carboxykinase [GTP], mitochondrial

sp|Q8BH04|PCKGM_MOUSE 3 0

950. Dedicator of cytokinesis protein 1 sp|Q8BUR4|DOCK1_MOUSE 2 0

951. Origin recognition complex subunit 4 sp|O88708|ORC4_MOUSE 6 0

952. Isoform 3 of ADP-dependent glucokinase

sp|Q8VDL4-3|ADPGK_MOUSE (+1)

2 0

953. Centromere/kinetochore protein zw10 homolog

sp|O54692|ZW10_MOUSE 2 0

954. Acyl-CoA dehydrogenase family member 9, mitochondrial

sp|Q8JZN5|ACAD9_MOUSE 3 0

955. Bystin sp|O54825|BYST_MOUSE 2 0

956. Beta-2 microglobulin tr|Q91XJ8|Q91XJ8_MOUSE 2 0

957. Glucose-6-phosphate 1-dehydrogenase X

sp|Q00612|G6PD1_MOUSE 6 0

958. Thioredoxin domain-containing protein 5

sp|Q91W90|TXND5_MOUSE 2 0


169

Table 0-2: Lipid species identified in control and TDM bead phagosomes. NAME Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 Pair 7

control [mol %]

TDM [mol %]

control [mol %]

TDM [mol %]

control [mol %]

TDM [mol %]

control [mol %]

TDM [mol %]

control [mol %]

TDM [mol %]

control [mol %]

TDM [mol %]

control [mol %]

TDM [mol %]

Cer [32:0] 2,69E-04 2,02E-04 2,12E-04 1,37E-03 4,03E-04 5,92E-04 2,42E-03

Cer [32:1] 6,03E-03 7,90E-03 3,35E-03 4,03E-03 2,45E-03 3,46E-03 4,14E-03 7,11E-03 3,72E-03 8,09E-03 4,29E-03 4,05E-03

Cer [34:0] 7,01E-02 5,62E-02 2,45E-01 2,50E-01 8,96E-03 2,80E-02 4,16E-02 1,48E-01 1,15E-02 5,86E-02 1,51E-02 6,64E-02 1,92E-02 1,59E-01


Cer [34:2] 4,53E-03 2,78E-03 2,72E-03 1,84E-03 2,74E-03 3,17E-03 4,88E-03 3,48E-03 6,39E-03 3,32E-03 2,98E-03

Cer [36:0] 3,39E-03 7,85E-03 1,15E-02 1,04E-01 5,00E-03 1,73E-02 7,13E-03 1,75E-02 7,39E-03 1,18E-01


Cer [36:2] 3,33E-03 1,78E-03 2,26E-03 1,82E-03 2,00E-03 2,50E-03 6,19E-03 2,74E-03 4,13E-03 3,71E-03 1,97E-03

Cer [37:0] 3,52E-03 5,53E-04 5,42E-04 2,55E-03 4,83E-03

Cer [40:1] 1,11E-01 1,61E-01 6,72E-02 5,36E-02 1,54E-01 1,22E-01 1,32E-01 1,79E-01 1,04E-01 4,61E-02




Cer [42:3] 2,63E-02 1,25E-02 1,39E-02 1,30E-02 2,11E-02 9,87E-03 1,21E-02 1,84E-02 1,19E-02 1,69E-02 2,13E-02 1,36E-02 7,70E-03

Cer [43:2] 3,35E-03 3,59E-03 4,21E-03 2,81E-03 2,31E-03 5,19E-03 5,95E-03 4,99E-03 6,24E-03 4,10E-03 3,19E-03

Cer [44:2] 4,53E-03 6,14E-03 3,56E-03 1,99E-03 4,55E-03 7,11E-03 4,42E-03 8,09E-03 3,97E-03 3,33E-03

Cholesterol 7,96E+00

1,28E+01

4,02E+00

6,47E+00

2,63E+01

2,82E+01

1,93E+01

2,74E+01

2,60E+01

3,04E+01

2,44E+01

2,64E+01

2,19E+01

3,50E+01

LBPA [32:1] 1,14E-02 6,82E-03 5,37E-03 4,95E-02 2,26E-02 6,21E-02 4,82E-02 5,63E-02 1,15E-01 6,81E-02 6,09E-02 7,94E-02 4,54E-02

LBPA [32:2] 1,87E-02 7,85E-03 1,26E-02 8,84E-03

LBPA [34:1] 7,82E-02 3,90E-02 2,46E-01 1,09E-01 2,61E-01 1,50E-01 5,10E-01 2,48E-01 2,86E-01 5,48E-01 3,50E-01 4,08E-01 4,45E-01 2,00E-01

LBPA [34:2] 1,83E-01 8,78E-02 6,36E-01 2,54E-01

LBPA [34:3] 1,69E-02 1,44E-01 6,74E-02

LBPA [34:4] 7,43E-03 2,86E-03 1,30E-03 3,52E-03 4,62E-03 6,19E-03 7,01E-03 6,20E-03 5,80E-03 9,52E-03 7,02E-03

LBPA [35:2] 2,61E-01 9,56E-02

LBPA [35:3] 2,28E-02 8,94E-03

LBPA [36:0] 3,49E-02 2,55E-02 1,87E-02 1,35E-02

LBPA [36:2] 6,47E+00

3,56E+00

2,89E+01

1,20E+01

6,05E+00

2,55E+00

1,91E+01

1,33E+01

1,07E+01

5,44E+00

1,27E+01

7,44E+00

1,59E+01

6,97E+00

LBPA [36:3] 2,96E+00

1,26E+00

5,01E-01 3,39E-01 2,08E+00

1,71E+00

1,17E+00

1,15E+00

1,21E+00

1,65E+00

1,84E+00

1,87E+00


170

LBPA [36:4] 4,04E-01 1,68E-01 5,37E-02 3,76E-02 1,73E-01 1,41E-01 1,46E-01 1,68E-01 1,49E-01 1,74E-01 2,21E-01 1,75E-01

LBPA [36:5] 3,35E-02 1,64E-02

LBPA [37:2] 5,37E-01 2,20E-01

LBPA [37:3] 3,23E-01 1,19E-01

LBPA [37:4] 6,81E-02 3,05E-02

LBPA [38:1] 4,99E-01 1,59E-01 2,79E-02 1,94E-01 8,81E-02 9,25E-02 1,01E-01 1,33E-01 1,68E-01 1,44E-01

LBPA [38:2] 1,29E+00

3,93E-01 1,27E-01 1,05E-01 1,26E+00

2,96E-01 1,59E-01 3,26E-01 1,81E-01 2,58E-01 3,04E-01 2,94E-01

LBPA [38:3] 2,42E+00

9,20E-01 1,21E-01 1,13E-01 1,26E+00

5,61E-01 2,68E-01 4,79E-01 2,79E-01 5,50E-01 4,96E-01 6,49E-01

LBPA [38:4] 2,80E+00

1,01E+00

2,08E-01 1,32E-01 8,61E-01 8,21E-01 5,19E-01 5,97E-01 4,99E-01 6,94E-01 7,90E-01 8,29E-01






LBPA [39:6] 3,19E-02 1,30E-02

LBPA [39:7] 1,04E-02

LBPA [39:9] 6,13E-03 2,79E-03 1,54E-02 4,13E-03 6,77E-03 7,79E-03 1,60E-02 6,33E-03

LBPA [40:1] 8,13E-03 3,64E-03


LBPA [40:4] 4,79E-01 1,19E-01 1,19E-02 3,45E-02 2,28E-02 3,75E-02 2,15E-02 3,12E-02 3,52E-02 3,23E-02

LBPA [40:5] 1,18E+00

3,75E-01 1,10E-01 2,99E-01 1,86E-01 2,88E-01 1,99E-01 2,86E-01 3,57E-01 3,17E-01

LBPA [40:6] 1,68E+00

5,39E-01 4,84E-01 2,85E-01 1,97E+00

1,37E+00

1,24E+00

1,35E+00

1,77E+00

1,61E+00

LBPA [40:7] 4,54E-01 1,02E+00

5,83E-01 3,11E+00

1,93E+00

2,63E+00

1,96E+00

1,97E+00

2,99E+00

2,40E+00

LBPA [40:8] 1,51E-02 2,84E-01 8,71E-02 9,86E-02 4,55E-02 1,99E-01 2,14E-01 2,76E-01 3,01E-01 2,49E-01 2,69E-01 4,21E-01 2,92E-01



LBPA [42:10] 4,74E-02 1,56E-02


LBPA [42:6] 9,78E-02 2,33E-02 2,83E-02 8,06E-02 6,71E-02 9,09E-02 7,39E-02 6,54E-02 1,05E-01 9,19E-02



171



LBPA [43:8] 2,98E-03 1,19E-03 4,83E-03 8,16E-03 1,08E-02 7,71E-03 9,91E-03 7,88E-03 1,72E-02 9,37E-03

LBPA [43:9] 9,23E-03

LBPA [44:10] 2,64E-02 1,23E-02 1,16E-01 6,54E-02 5,87E-02 2,14E-02 1,01E-01 1,33E-01 2,08E-01 1,33E-01 2,04E-01 1,40E-01 2,91E-01 1,51E-01

LBPA [44:7] 5,36E-03 5,14E-03 1,27E-03 3,97E-03 3,76E-03 4,17E-03 5,37E-03 4,87E-03


LPC [16:1] 3,70E-02 5,16E-02 1,56E-02 8,89E-03 9,85E-03 1,49E-02 1,36E-02 2,31E-02 1,78E-02 1,31E-02 1,76E-02 2,36E-02

LPC [18:1] 2,92E-01 3,17E-01 2,72E-01 3,34E-01 8,22E-02 5,15E-02 1,04E-01 1,17E-01 1,13E-01 2,67E-01 1,62E-01 1,29E-01 1,24E-01 1,91E-01


LPC [18:3] 2,46E-01 1,45E-02 1,06E-02 4,82E-03 9,24E-03 1,32E-02 5,87E-03 1,83E-02 1,36E-02 1,60E-02 1,66E-02


PC [28:0] 1,82E-01 2,95E-01 1,01E-01 7,79E-02 1,34E-01 1,17E-01 1,24E-01 8,95E-02 8,00E-02 6,59E-02

PC [30:0] 2,10E+00

2,08E+00

1,22E+00

1,39E+00

1,81E+00

2,58E+00

1,14E+00

9,48E-01 1,51E+00

1,26E+00

1,35E+00

1,21E+00

1,04E+00

7,82E-01

PC [30:1] 3,43E-01 3,07E-01 2,61E-01 3,12E-01 2,35E-01 3,11E-01 1,39E-01 1,49E-01 2,04E-01 1,68E-01 1,77E-01 1,56E-01 1,42E-01 1,35E-01

PC [30:3] 9,12E-03 8,31E-03 6,89E-03 3,53E-03 6,84E-03 7,94E-03 9,69E-03 6,15E-03 2,67E-03 3,57E-03

PC [32:0] 3,63E+00

3,69E+00

1,53E+00

1,93E+00

1,68E+00

2,01E+00

1,06E+00

1,49E+00

1,52E+00

1,18E+00

1,29E+00

1,36E+00

1,20E+00

1,09E+00

PC [32:1] 4,57E+00

4,29E+00

2,38E+00

2,80E+00

3,73E+00

4,54E+00

2,17E+00

1,92E+00

2,83E+00

2,38E+00

2,54E+00

2,34E+00

1,97E+00

1,70E+00



PC [32:4] 1,29E-02 8,45E-03 7,94E-03 1,03E-02 8,71E-03 1,17E-02 1,41E-02 7,46E-03 1,00E-02 8,68E-03




PC [33:7] 1,37E-02 9,64E-03 8,88E-03 8,67E-03 9,94E-03 1,30E-02 1,20E-02 1,38E-02 1,08E-02 8,92E-03

PC [34:1] 1,17E+01

1,10E+01

3,91E+00

6,40E+00

8,30E+00

7,98E+00

5,26E+00

4,59E+00

7,44E+00

5,48E+00

6,75E+00

6,92E+00

5,55E+00

3,68E+00

PC [34:2] 3,23E+00

2,81E+00

1,65E+00

2,41E+00

2,58E+00

2,61E+00

1,55E+00

1,81E+00

3,00E+00

1,69E+00

2,57E+00

2,46E+00

2,02E+00

1,57E+00





172


PC [36:1] 1,75E+00

1,50E+00

5,35E-01 1,34E+00

8,92E-01 8,96E-01 6,44E-01 9,54E-01 5,22E-01 8,83E-01 5,92E-01 6,19E-01 5,29E-01 8,13E-01

PC [36:2] 4,90E+00

4,14E+00

3,30E+00

6,17E+00

4,28E+00

4,62E+00

3,24E+00

4,19E+00

4,82E+00

3,44E+00

4,62E+00

4,61E+00

3,83E+00

3,83E+00

PC [36:3] 9,98E-01 8,56E-01 7,12E-01 1,14E+00

9,52E-01 1,08E+00

5,91E-01 1,01E+00

1,56E+00

6,70E-01 1,41E+00

1,20E+00

1,05E+00

9,32E-01



PC [37:1] 7,51E-02 4,94E-03 7,38E-03 6,38E-03 4,89E-03 7,26E-03 4,83E-03 5,26E-03 3,22E-03 4,04E-03

PC [37:2] 8,01E-02 3,06E-02 7,35E-02 3,69E-02 3,63E-02 2,86E-02 3,02E-02 3,57E-02 3,29E-02 3,42E-02 3,21E-02 2,76E-02 2,76E-02

PC [38:1] 1,40E-02 1,61E-02 1,14E-02 6,63E-03 7,48E-03 1,26E-02 7,60E-03 8,57E-03 5,25E-03 5,15E-03






PC [40:2] 5,85E-02 9,36E-03 1,35E-02 1,90E-02 1,26E-02 6,04E-03 6,17E-03 7,96E-03 5,11E-03 5,36E-03 6,60E-03 5,89E-03

PC [40:3] 1,44E-02 1,46E-02 9,53E-03 1,35E-02 6,44E-03 4,55E-03 9,51E-03 8,02E-03 8,58E-03 7,14E-03 6,52E-03 3,94E-03

PC [40:4] 6,20E-02 2,26E-02 2,72E-02 1,00E-02 1,24E-02 6,28E-03 4,68E-03 9,39E-03 6,72E-03 5,30E-03 4,43E-03 5,36E-03

PC [40:5] 4,04E-02 4,37E-02 1,65E-02 2,54E-02 1,13E-02 1,02E-02 1,78E-02 1,08E-02 1,60E-02 1,09E-02 1,13E-02 8,55E-03

PC [40:7] 2,37E-02 2,21E-02 1,85E-02 2,87E-02 9,58E-03 1,17E-02 2,04E-02 8,56E-03 2,07E-02 1,08E-02 1,28E-02 1,32E-02

PC [44:10] 8,40E-03 1,56E-02 2,79E-03 3,00E-03 5,17E-03 4,22E-03 3,08E-03 5,44E-03 1,51E-03 3,26E-03

PC-O [28:0] 4,80E-02 2,76E-02 2,91E-02 2,64E-02 4,03E-02 1,91E-02 1,66E-02 2,33E-02 1,61E-02 2,18E-02 2,37E-02 1,47E-02 1,76E-02

PC-O [29:0] 5,51E-02 4,03E-02 2,80E-02 2,35E-02 2,03E-02 2,58E-02 1,60E-02 1,03E-02 1,37E-02 1,51E-02 1,33E-02 1,15E-02 9,61E-03 9,44E-03

PC-O [30:0] 1,12E+00

9,76E-01 5,19E-01 6,46E-01 4,08E-01 4,38E-01 4,19E-01 2,70E-01 2,17E-01 2,54E-01 2,12E-01 2,15E-01 1,89E-01 2,05E-01




PC-O [31:2] 1,78E-02 9,36E-03 1,24E-02 8,64E-03 8,25E-03 9,72E-03 6,73E-03 1,08E-02 1,11E-02 9,61E-03 8,34E-03

PC-O [32:0] 5,34E-01 5,15E-01 5,86E-01 5,81E-01 2,49E-01 4,71E-01 2,66E-01 3,21E-01 2,78E-01 4,26E-01

PC-O [32:2] 5,62E-02 7,36E-02 4,37E-02 4,98E-02 2,86E-02 3,05E-02 4,30E-02 2,90E-02 3,61E-02 3,04E-02 3,29E-02 2,62E-02

PC-O [32:3] 1,84E-02 1,16E-02 1,88E-02 1,15E-02 8,85E-03 1,35E-02 1,60E-02 1,28E-02 1,31E-02 1,27E-02



173



PC-O [33:3] 1,01E-02 1,17E-02 9,68E-03 7,58E-03 6,54E-03 6,75E-03 1,01E-02 1,14E-02 1,06E-02 7,78E-03 9,48E-03

PC-O [34:1] 5,30E+00

4,07E+00

2,11E+00

3,27E+00

2,17E+00

2,09E+00

2,13E+00

1,87E+00

1,43E+00

1,27E+00

1,36E+00

1,48E+00

1,24E+00

1,38E+00




PC-O [35:3] 1,14E-02 8,93E-03 8,52E-03 5,91E-03 7,39E-03 8,55E-03 5,65E-03 7,01E-03 7,52E-03 5,44E-03 5,67E-03

PC-O [35:4] 7,96E-03 5,32E-03 5,95E-03 5,82E-03 7,10E-03 7,96E-03 9,03E-03 6,06E-03 7,35E-03 5,72E-03


PC-O [36:2] 1,04E+00

7,53E-01 1,07E+00

1,38E+00

9,09E-01 9,80E-01 8,04E-01 8,29E-01 6,88E-01 4,61E-01 6,36E-01 5,28E-01 5,05E-01 5,77E-01




PC-O [37:3] 2,16E-02 3,75E-02

PC-O [37:6] 1,51E-02 1,87E-02 8,76E-03 1,36E-02 2,02E-02 2,07E-02 1,58E-02 1,91E-02 2,39E-02 2,03E-02 1,27E-02

PC-O [38:1] 6,35E-02 4,54E-03 2,03E-02 7,00E-03 6,75E-03 8,43E-03 4,97E-03 3,36E-03 4,94E-03

PC-O [38:2] 3,76E-02 4,95E-02 1,27E-02 9,56E-03 1,51E-02 8,37E-03 6,03E-03 9,16E-03 9,20E-03 8,28E-03 8,96E-03 7,22E-03

PC-O [38:3] 2,74E-02 3,21E-02 2,49E-02 3,03E-02 2,48E-02 1,29E-02 2,22E-02 2,22E-02 1,99E-02 2,29E-02


PC-O [40:2] 1,18E-02 2,28E-02 4,59E-03 4,96E-03 3,61E-03 3,97E-03

PC-O [40:5] 2,49E-02 2,21E-02 4,55E-03 6,47E-03 4,43E-03 2,33E-03 2,85E-03 3,33E-03 3,56E-03

PC-O [40:6] 7,10E-02 5,93E-02 1,29E-02 1,85E-02 1,09E-02 7,40E-03 9,64E-03 7,70E-03 5,88E-03 7,07E-03

PE [30:0] 6,22E-03 6,33E-03 6,52E-03 9,99E-03 7,39E-03 9,09E-03 9,91E-03 4,97E-03 8,17E-03 6,60E-03 5,52E-03 6,78E-03

PE [32:0] 1,37E-02 1,53E-02 1,44E-02 2,31E-02 1,69E-02 2,52E-02 1,89E-02 9,86E-03 1,74E-02 1,35E-02 1,19E-02 1,81E-02

PE [32:1] 1,14E-01 9,21E-02 9,88E-02 1,26E-01 8,82E-02 1,17E-01 7,11E-02 6,27E-02 6,40E-02 5,12E-02 5,98E-02 4,90E-02 4,44E-02 6,05E-02

PE [32:2] 8,62E-03 9,02E-03 5,83E-03 8,29E-03 7,67E-03 6,63E-03 6,75E-03 7,66E-03 6,17E-03 7,60E-03

PE [33:0] 2,83E-03 3,77E-03 2,37E-03 3,42E-03 3,62E-03 3,11E-03 3,18E-03 2,93E-03 2,96E-03




PE [34:3] 1,80E-02 3,03E-02 3,82E-02 3,11E-02 2,17E-02 2,27E-02 2,20E-02 1,61E-02 2,29E-02 2,16E-02 1,92E-02 2,24E-02


174




PE [36:2] 5,58E-01 1,09E+00

8,22E-01 1,74E+00

8,95E-01 6,25E-01 6,63E-01 1,03E+00

1,41E+00

7,71E-01 1,42E+00

1,14E+00

1,11E+00

1,04E+00



PE [36:5] 6,69E-03 1,60E-02 1,05E-02 9,62E-03 1,19E-02 1,52E-02 1,32E-02 1,47E-02 1,32E-02 1,47E-02 1,46E-02

PE [37:1] 2,36E-03 3,89E-03 2,65E-03 1,84E-03 1,81E-03 2,93E-03 2,57E-03 2,02E-03 2,67E-03 2,07E-03 2,04E-03 3,85E-03

PE [37:2] 7,61E-03 1,97E-02 9,81E-03 6,37E-03 8,53E-03 1,19E-02 1,64E-02 1,11E-02 1,72E-02 1,46E-02 1,37E-02 1,47E-02

PE [37:3] 5,72E-03 1,34E-02 5,96E-03 4,60E-03 4,45E-03 8,16E-03 1,02E-02 5,66E-03 9,33E-03 9,64E-03 8,06E-03 8,79E-03

PE [38:1] 2,73E-03 8,95E-03 4,31E-03 2,87E-03 2,63E-03 3,10E-03 3,46E-03 3,55E-03 3,10E-03 3,57E-03 3,24E-03 3,17E-03

PE [38:2] 1,50E-02 3,11E-03 5,69E-03 3,79E-03 4,46E-03 1,19E-02 1,00E-02 7,85E-03 1,20E-02 1,12E-02 9,72E-03 7,57E-03 1,06E-02




PE [38:6] 2,91E-02 1,12E-02 2,46E-02 4,04E-02 3,00E-02 2,13E-02 2,10E-02 3,59E-02 2,30E-02 3,24E-02 2,68E-02 2,89E-02 2,83E-02

PE [40:2] 1,96E-03 1,63E-03 2,48E-03 1,72E-03 1,60E-03 1,64E-03 1,71E-03 2,20E-03 1,44E-03

PE [40:4] 1,98E-03 1,85E-03 2,07E-03 2,07E-03 3,73E-03 2,46E-03 2,91E-03 4,07E-03 2,90E-03 2,03E-03



PE [40:7] 8,67E-03 1,76E-02 1,38E-02 1,35E-02 9,82E-03 6,95E-03 1,26E-02 9,30E-03 1,20E-02 9,80E-03 8,93E-03 9,92E-03

PE-O [30:0] 1,00E-03 1,35E-03 1,14E-03 1,59E-03 1,25E-03 2,06E-03 1,37E-03 1,69E-03 2,01E-03

PE-O [30:1] 2,53E-03 2,59E-03 2,90E-03 4,44E-03 4,72E-03 3,44E-03 3,54E-03 2,68E-03 2,42E-03 2,62E-03 4,58E-03

PE-O [32:0] 6,66E-03 1,69E-02 3,32E-03 3,42E-03 3,15E-03 4,71E-03 1,46E-03 4,03E-03 2,03E-03 3,23E-03 1,87E-03 4,15E-03

PE-O [32:1] 2,21E-02 9,96E-03 1,01E-02 1,51E-02 2,17E-02 1,42E-02 1,30E-02 1,09E-02 1,57E-02

PE-O [32:2] 3,25E-02 1,92E-02 6,11E-02 7,69E-02 4,72E-02 4,80E-02 6,06E-02 6,07E-02 4,34E-02 6,90E-02 4,36E-02 4,90E-02 4,12E-02 6,66E-02

PE-O [32:3] 3,95E-03 2,45E-03 2,03E-03 2,72E-03 3,86E-03 3,40E-03 5,08E-03 3,51E-03 4,59E-03 3,13E-03 4,85E-03


PE-O [33:2] 2,69E-02 1,32E-02 2,06E-02 1,62E-02 1,37E-02 1,73E-02 1,99E-02 1,78E-02 2,49E-02 1,73E-02 1,94E-02 1,61E-02 2,44E-02

PE-O [33:3] 2,86E-03 3,25E-03 2,48E-03 3,00E-03 2,76E-03 4,37E-03 6,05E-03 4,66E-03 3,99E-03 3,89E-03 4,37E-03

PE-O [34:0] 5,38E-03 4,51E-03 3,32E-03 4,05E-03 8,37E-03 4,24E-03 5,80E-03 3,79E-03 4,09E-03 2,46E-03 6,79E-03




175



PE-O [35:3] 3,66E-02 2,32E-02 3,86E-02 4,93E-02 5,27E-02 7,77E-02 5,65E-02 5,64E-02 5,57E-02 6,18E-02







PE-O [37:3] 4,75E-03 2,22E-03 4,88E-03 6,96E-03 4,95E-03 9,69E-03 5,68E-03 8,06E-03 6,03E-03 9,70E-03















PE-O [40:7] 1,27E-01 1,26E-01 1,21E-01 4,27E-02 1,10E-01 7,56E-02 8,98E-02 6,56E-02 7,31E-02 7,52E-02

PE-O [42:7] 2,63E-03 1,85E-03 2,13E-03 1,35E-03 3,27E-03 3,08E-03 3,48E-03

PG [32:0] 9,92E-03 7,95E-03 6,96E-03 5,95E-03 8,88E-03 1,05E-02 9,08E-03 9,16E-03 7,96E-03 8,18E-03

PG [32:2] 1,76E-03 2,62E-03 9,04E-04 8,29E-04 8,34E-04 6,80E-04 7,83E-04 9,40E-04 1,90E-03 5,87E-04 7,52E-04 8,92E-04

PG [33:2] 7,56E-04 2,49E-04 1,66E-04 3,28E-04 8,16E-04

PG [34:0] 2,92E-03 1,85E-02 1,59E-02 1,97E-02 1,88E-02 2,63E-02 1,65E-02 2,28E-02 2,20E-02 2,61E-02

PG [34:1] 2,62E-01 1,78E-01 1,32E-01 9,72E-02 4,05E-01 5,19E-01 1,90E-01 6,22E-02 2,33E-01 1,04E-01 2,35E-01 1,44E-01 1,18E-01 4,73E-02


PG [34:3] 1,16E-02 9,70E-03 1,01E-02 2,37E-03 2,28E-03 2,80E-03 3,14E-03 3,57E-03 1,72E-03 8,39E-03 2,17E-03 2,42E-03 3,48E-03


176



PG [35:3] 4,81E-03 1,56E-03 1,94E-03 4,22E-04 2,97E-04 6,44E-04 4,18E-04 6,12E-04 1,86E-03 5,79E-04 6,98E-04





PG [36:5] 6,57E-03 2,01E-03 2,31E-03 5,53E-04 3,63E-04 8,97E-04 1,03E-03 2,82E-03 7,35E-04

PG [37:1] 1,50E-03 2,07E-03 1,12E-03 4,97E-04 1,11E-03 1,40E-03 1,13E-03 6,71E-04

PG [37:3] 8,21E-03 5,52E-03 7,10E-03 7,21E-04 5,37E-04 1,74E-03 9,89E-04 1,31E-03 4,98E-03 8,76E-04 1,14E-03 2,24E-03

PG [37:4] 2,16E-03 2,20E-04 5,84E-04 4,28E-04 4,49E-04 1,86E-03 5,55E-04






PG [38:6] 1,01E-02 1,24E-02 1,45E-03 8,88E-04 3,51E-03 1,59E-03 3,38E-03 1,28E-02 2,48E-03 3,60E-03

PG [39:3] 2,20E-03 2,72E-03 5,61E-04 1,35E-03

PG [39:4] 6,83E-03 4,53E-03 4,81E-03 9,06E-04 4,49E-04 8,39E-04 2,71E-03 5,93E-04 1,14E-03

PG [40:3] 1,61E-03 1,81E-03 2,72E-04 8,87E-04 3,70E-04 5,12E-04 2,11E-03 4,97E-04 8,87E-04

PG [40:4] 5,97E-03 4,88E-03 6,16E-03 3,40E-04 1,97E-04 1,28E-03 5,45E-04 1,00E-03 3,58E-03 6,45E-04 8,80E-04 1,32E-03



PG [42:6] 1,47E-03 5,44E-04 6,68E-04 2,99E-03

PI [34:2] 7,43E-02 6,93E-02 1,67E-02 2,34E-02 6,84E-02 8,85E-02 2,95E-02 1,50E-02 4,10E-02 7,95E-02 4,43E-02 4,76E-02 3,87E-02 1,43E-02

PI [35:1] 2,96E-02 1,87E-02 4,28E-03 1,97E-02 2,59E-02 7,13E-03 3,67E-03 8,21E-03 1,82E-02 7,82E-03 1,03E-02 7,52E-03 2,61E-03



PI [36:2] 9,47E-01 8,76E-01 3,98E-01 6,04E-01 1,59E+00

2,19E+00

8,41E-01 4,68E-01 9,79E-01 1,26E+00

9,32E-01 9,27E-01 7,73E-01 4,85E-01


PI [36:4] 1,24E-02 5,20E-03 3,14E-02 4,77E-02 2,10E-02 7,32E-03 2,83E-02 1,44E-02 2,24E-02 1,63E-02 1,58E-02 6,43E-03




177

PI [37:4] 2,88E-02 2,34E-02 1,10E-02 1,48E-02

PI [38:2] 3,55E-02 3,91E-02 9,63E-04 7,19E-02 8,26E-03 2,10E-02

PI [38:3] 1,19E+00

1,06E+00

2,24E+00

3,60E+00

2,36E+00

2,73E+00

1,71E+00

1,95E+00

2,13E+00

1,37E+00

2,11E+00

1,53E+00

1,45E+00

1,83E+00

PI [38:4] 1,07E+00

9,83E-01 1,50E+00

2,31E+00

1,57E+00

2,31E+00

1,28E+00

1,08E+00

1,84E+00

1,01E+00

1,54E+00

1,12E+00

1,16E+00

1,20E+00


PI [38:6] 6,24E-03 9,82E-03 1,28E-02 1,86E-02 9,41E-03 3,95E-03 1,61E-02 1,78E-02 1,38E-02 1,03E-02 1,08E-02 4,89E-03



PI [39:5] 3,08E-03 4,95E-03 6,03E-03 3,49E-03 2,29E-03 5,95E-03 4,99E-03 5,92E-03 5,21E-03 4,16E-03 2,66E-03




PI [40:6] 2,25E-02 1,59E-02 2,44E-02 5,04E-02 3,46E-02 1,38E-02 5,67E-02 4,46E-02 4,66E-02 2,77E-02 2,63E-02 1,64E-02

PI [40:7] 7,29E-03 1,17E-02 9,52E-03 9,68E-03 5,78E-03 4,90E-03 1,88E-02 8,15E-03 1,30E-02 7,11E-03 8,95E-03 7,33E-03

PS [33:1] 1,52E-02 1,73E-02 1,22E-02 1,39E-02 1,42E-02 2,13E-02 2,95E-02

PS [34:1] 7,64E-01 6,88E-01 2,53E-01 3,25E-01 6,47E-01 7,86E-01 5,17E-01 3,80E-01 3,66E-01 6,21E-01 3,42E-01 4,64E-01 3,68E-01 3,41E-01

PS [34:2] 2,19E-02 6,38E-02 7,76E-02 5,78E-02 4,78E-02 5,04E-02 8,90E-02 4,58E-02 6,48E-02 5,40E-02 6,06E-02

PS [35:1] 1,40E-01 1,67E-01 2,43E-01 1,15E-01 1,36E-01 1,14E-01 1,75E-01 1,27E-01 2,19E-01 5,70E-01 1,36E-01 1,48E-01 2,51E-01

PS [35:2] 2,19E-02 3,43E-02 1,55E-02 1,98E-02 1,92E-02 2,45E-02 1,66E-02 5,19E-02 3,58E-02 2,98E-02 4,01E-02

PS [36:1] 4,07E+00

3,34E+00

2,19E+00

2,78E+00

2,64E+00

3,07E+00

2,25E+00

2,26E+00

1,99E+00

2,62E+00

1,82E+00

2,44E+00

2,08E+00

1,89E+00

PS [36:2] 1,18E+00

8,80E-01 5,65E-01 6,41E-01 1,04E+00

1,31E+00

1,05E+00

9,43E-01 9,72E-01 1,25E+00

8,85E-01 1,15E+00

1,03E+00

8,38E-01

PS [36:4] 9,80E-03 3,42E-02 4,24E-02 2,42E-02 1,98E-02 3,03E-02 2,22E-02


PS [37:2] 1,95E-01 2,55E-01 3,06E-02 3,09E-02 4,59E-02 1,48E-01 6,64E-02 9,17E-02 5,79E-02 2,28E-01

PS [37:3] 2,27E-02 5,00E-02 5,13E-03 9,83E-03 1,76E-02 5,60E-02 2,91E-02

PS [38:0] 4,72E-02 1,13E-02 2,48E-02 4,89E-02 3,32E-02 8,75E-02 6,09E-02 3,94E-02 9,45E-02

PS [38:1] 1,82E-02 3,06E-02 3,71E-02 3,31E-02 2,45E-02 3,03E-02 2,00E-02 7,01E-02 3,48E-02 2,49E-02 4,03E-02 2,15E-02

PS [38:2] 1,67E-02 3,21E-02 3,36E-02 3,73E-02 3,78E-02 2,85E-02 2,62E-02 4,01E-02 2,68E-02 2,53E-02 2,51E-02 2,33E-02



PS [38:5] 2,81E-02 3,71E-02 5,11E-02 6,65E-02 5,39E-02 5,33E-02 7,27E-02 9,81E-02 6,20E-02 7,53E-02 6,01E-02 7,45E-02


178

PS [39:1] 8,24E-03 3,58E-02 8,71E-03 4,39E-03 6,77E-03 1,87E-02 1,31E-02 4,91E-02 4,55E-02 4,47E-02 1,80E-02 3,92E-02

PS [39:3] 6,20E-02 7,84E-02 5,41E-03 3,33E-03 7,52E-03 2,47E-02 1,17E-02 5,00E-02 1,79E-02 2,21E-02 2,76E-02

PS [39:4] 1,47E-01 1,77E-01 1,08E-02 7,22E-03 1,81E-02 7,04E-02 4,74E-02 4,68E-02 3,66E-02 3,01E-02 4,05E-02 1,19E-01

PS [40:1] 4,39E-02 2,25E-02 3,04E-02 5,80E-02 3,03E-02 8,18E-02 5,54E-02 2,83E-02 8,73E-02

PS [40:2] 6,17E-03 2,44E-02 2,05E-02 1,98E-02 1,88E-02 1,88E-02 2,18E-02 2,69E-02

PS [40:3] 2,49E-02 3,85E-02 1,17E-02 9,06E-03 1,43E-02 1,63E-02 1,82E-02 1,51E-02

PS [40:4] 1,03E-02 5,89E-03 4,62E-03 7,66E-03

PS [40:5] 1,14E-01 3,84E-02 4,66E-02 1,85E-02 1,61E-02 2,62E-02 1,24E-02 1,57E-02 2,08E-02 2,46E-02


PS [43:6] 1,73E-02 1,02E-02 1,34E-02 1,48E-02 2,47E-02 2,53E-02 2,78E-02 2,80E-02 2,80E-02

SM [32:1] 2,53E-01 2,95E-01 9,19E-02 1,55E-01 7,94E-02 6,95E-02 6,44E-02 6,78E-02 6,16E-02 1,03E-01 6,08E-02 8,25E-02 8,11E-02 7,50E-02


SM [34:1] 1,14E+01

1,32E+01

3,73E+00

7,08E+00

4,85E+00

4,18E+00

4,20E+00

4,31E+00

3,45E+00

5,23E+00

3,25E+00

5,02E+00

4,71E+00

4,29E+00

SM [34:4] 1,24E-02 1,89E-02 9,72E-03 1,31E-02 1,28E-02 1,12E-02 2,19E-02 1,32E-02 1,64E-02 1,47E-02 1,36E-02


SM [35:4] 1,50E-02 7,63E-03 1,25E-02 1,15E-02 8,77E-03 1,54E-02 1,33E-02 1,20E-02 1,65E-02 8,55E-03

SM [36:1] 1,01E+00

1,08E+00

3,96E-01 6,60E-01 4,13E-01 3,81E-01 3,53E-01 4,17E-01 5,29E-01 6,26E-01 4,26E-01 7,06E-01 6,63E-01 4,05E-01

SM [36:4] 6,95E-01 1,16E+00

3,05E-01 7,34E-01 6,86E-01 3,62E-01 5,59E-01 5,32E-01 3,62E-01 7,18E-01 4,48E-01 4,91E-01 4,96E-01 4,47E-01

SM [36:5] 7,49E-03 4,13E-03 6,43E-03 5,71E-03 3,72E-03 1,01E-02 7,02E-03 7,11E-03 8,85E-03 8,93E-03

SM [37:1] 6,77E-03 8,03E-03 8,49E-03 6,71E-03 1,14E-02 1,21E-02 1,15E-02 1,41E-02 1,34E-02 8,11E-03

SM [37:4] 1,03E-02 4,39E-03 8,05E-03 8,35E-03 4,99E-03 1,41E-02 8,07E-03 8,81E-03 7,82E-03 9,89E-03


SM [38:4] 2,92E-02 7,45E-02

SM [40:1] 1,20E+00

1,62E+00

3,63E-01 6,77E-01 4,49E-01 4,78E-01 3,81E-01 3,66E-01 3,79E-01 6,53E-01 3,14E-01 5,70E-01 4,87E-01 5,09E-01


SM [40:4] 1,56E-02 4,76E-03 1,12E-02 7,60E-03 7,70E-03 1,78E-02 1,30E-02 1,22E-02 1,09E-02 1,08E-02


SM [41:2] 1,03E-01 4,08E-02 8,15E-02 4,84E-02 4,40E-02 4,64E-02 4,77E-02 4,25E-02 7,40E-02 3,76E-02 5,76E-02 5,54E-02 5,54E-02

SM [42:1] 8,04E-01 1,05E+00

2,18E-01 4,23E-01

SM [42:2] 2,65E+00

2,91E+00

1,32E+00

2,40E+00

1,42E+00

1,62E+00

1,69E+00

1,47E+00

1,16E+00

1,78E+00

1,06E+00

1,85E+00

1,61E+00

1,81E+00


179


SM [42:4] 5,57E-02 2,29E-02 4,23E-02 3,68E-02 3,10E-02 7,36E-02 3,98E-02 5,81E-02 4,28E-02 5,55E-02

SM [43:1] 5,81E-03 4,22E-03 6,13E-03 3,63E-03 1,01E-02 3,21E-03 5,83E-03 3,79E-03 5,93E-03

SM [43:2] 7,58E-02 9,99E-03 1,46E-02 1,17E-02 9,91E-03 1,07E-02 8,33E-03 1,47E-02 8,80E-03 9,81E-03 9,28E-03 1,21E-02

SM [44:5] 2,09E-01 2,01E-01 1,09E-01 2,02E-01 1,81E-01 5,70E-02 1,50E-01

List of figures

180

List of figures

Figure 1.1: Global trends in estimated rates of TB incidence in 2013.. .................... 12

Figure 1.2: Stages of M. tuberculosis infection.. ...................................................... 12

Figure 1.3: The immunological process following M. tuberculosis infection in the lung..

................................................................................................................................ 14

Figure 1.4: Signalling events leading to actin polymerization during FcγR–mediated

phagocytosis.. .......................................................................................................... 19

Figure 1.5: Stages of phagosome maturation. ......................................................... 21

Figure 1.6: Production of ROI and RNI in the phagosome. ...................................... 23

Figure 1.7: Schematic representation of the composition of the mycobacterial cell-wall.

................................................................................................................................ 27

Figure 1.8: Acyltrehaloses TMM and TDM of the mycobacterial outer membrane. .. 28

Figure 3.1: Scheme of the procedure for isolation and purification of bead phagosomes

from macrophages for mass spectrometry analysis.. ............................................... 55

Figure 3.2: β-galactosidase assay of purified control versus TDM bead phagosomes.

................................................................................................................................ 56

Figure 3.3: Control and TDM bead phagosomes prior to and after sorting by FACS..

................................................................................................................................ 57

Figure 3.4: Intersection diagram of total numbers of identified proteins in proteomes of

control versus TDM bead phagosome samples. ...................................................... 58

Figure 3.5: Identified proteins in control and TDM beads phagosomes grouped

according to their cellular localization. ..................................................................... 59

Figure 3.6: Percentage distribution of all proteins identified in control versus TDM bead

phagosome samples grouped according to their cellular localization. ...................... 60

Figure 3.7: STRING network of proteins enriched in TDM bead phagosomes.. ....... 67

Figure 3.8: Immunofluorescence staining of control and TDM bead infected RAW264.7

macrophages for annexinA1 and LAMP1. ............................................................... 70

Figure 3.9: Immunofluorescence staining of control, TDM bead and M. tuberculosis

GFP infected RAW264.7 macrophages for annexinA6 and LAMP1......................... 72


GFP infected RAW264.7 macrophages for cofilin1 and LAMP1. ............................. 74


GFP infected RAW264.7 macrophages for profilin1 and LAMP1. ............................ 76


GFP infected RAW264.7 macrophages for SNAP23 and LAMP1. ........................... 78

List of figures

181


GFP infected RAW264.7 macrophages for VAMP3 and LAMP1. ............................. 80

Figure 3.14: Quantification of candidate proteins annexinA1, annexinA6, profilin1,

SNAP23 and VAMP3 on isolated control versus TDM bead phagosomes via Western

blot. .......................................................................................................................... 81

Figure 3.15: Control of siRNA-mediated knock-down of candidate proteins annexinA6,

cofilin1, profilin1 and VAMP3 via Western blot. ........................................................ 82

Figure 3.16: Survival of M. tuberculosis inside RAW264.7 macrophages after knock-

down of annexinA6, cofilin1, profilin1 and VAMP3 using RNA interference.............. 83

Figure 3.17: Accumulation of β-actin around control and TDM bead phagosomes in

RAW264.7 macrophages. ........................................................................................ 84

Figure 3.18: Localization of β-actin and WASH1 around M. tuberculosis GFP

phagosomes in RAW264.7 macrophages. ............................................................... 85

Figure 3.19: Co-localization of lysotracker and LAMP1 with M. tuberculosis GFP in

RAW264.7 macrophages after removal of β-actin with LatrunkulinA (LatA). ............ 86

Figure 3.20: Survival of M. tuberculosis GFP in RAW264.7 macrophages after removal

of β-actin with LatrunkulinA (LatA). .......................................................................... 86

Figure 3.21: Control of siRNA-mediated knock-down of WASH1 via Western blot.. . 87

Figure 3.22: Survival of M. tuberculosis inside RAW264.7 macrophages after knock-

down of WASH1 using RNA interference. ................................................................ 88

Figure 3.23: β-galactosidase activity of all seven paired samples used for lipidomic

analysis of bead phagosomes. ................................................................................. 89

Figure 3.24: Identified lipid classes in control bead phagosomes. ............................ 90

Figure 3.25: Before-and-after-plot of Cer, Chol, LBPA, LPC, PC and PC-O identified

in control versus TDM bead phagosomes.. .............................................................. 91

Figure 3.26: Before-and-after-plot of PE, PE-O, PG, PI, PS and SM identified in control

versus TDM bead phagosomes. ............................................................................. 92

Figure 3.27: Volcano-plot - Distribution of identified lipid species in control versus TDM

bead phagosome samples.94

Figure 4.1: Scheme of the potential function of TDM-mediated inhibition of phagosome

maturation. ............................................................................................................. 121

List of tables

182

List of tables

Table 2-1: Consumables .......................................................................................... 31

Table 2-2: Chemicals ............................................................................................... 32

Table 2-3: siRNA ..................................................................................................... 34

Table 2-4: Lipids ...................................................................................................... 34

Table 2-5: Antibodies and dyes ............................................................................... 35

Table 2-6: Cell-lines ................................................................................................. 35

Table 2-7: Bacteria .................................................................................................. 35

Table 2-8: Hardware ................................................................................................ 35

Table 2-9: Software ................................................................................................. 37

Table 2-10: Elution gradient of peptides with buffer B. ............................................. 43

Table 2-11: Dilutions of primary and secondary antibodies and other labels ........... 46

Table 2-12: SDS-PAGE gel formulations. ................................................................ 47

Table 2-13: Concentrations of antibodies used for Western blot. ............................. 47

Table 2-14: Internal standard mix ............................................................................ 49

Table 2-15: Gradient and flow rates used for the separation of phospholipids by µLC-

FT-ICR-MS .............................................................................................................. 50

Table 2-16: FT-ICR-MS instrumental settings .......................................................... 51

Table 2-17: Flow rates used for the analysis of cholesteryl acetate by ESI Qq-TOF-MS

................................................................................................................................ 52

Table 3-1: List of proteins enriched in TDM bead phagosomes compared to controls..

................................................................................................................................ 61

Table 3-2: List of proteins enriched in control bead phagosomes. ........................... 62

Table 3-3: Cellular localization and biological process of proteins enriched to TDM

bead over control phagosomes. ............................................................................... 62

Table 3-4: Identified proteins selected for further analysis as potential host-cell derived

TDM interaction partners. ........................................................................................ 68

Table 4-1: Comparison of proteins identified in BCG versus TDM bead phagosomes..

.............................................................................................................................. 103

Table 4-2: Comparison of proteins identified in ManLAM versus TDM bead

phagosomes.. ........................................................................................................ 105

Table 0-1: List of all proteins identified in control and TDM bead phagosomes. .... 141

Table 0-2: Lipid species identified in control and TDM bead phagosomes. ........... 169

List of abbreviations and symbols

183


M. tuberculosis Mycobacterium tuberculosis

TDM trehalose-6,6´dimycolate

TB tuberculosis

WHO world health organization

MTBC Mycobacterium tuberculosis complex

M. bovis Mycobacterium bovis

BCG bacille Calmette-Guérin

HIV human immunodeficiency virus

AIDS acquired immunodeficiency syndrome

MDR TB multi drug resistant tuberculosis

XDR TB extensively drug resistant tuberculosis

TNF-α tumor necrosis factor alpha

DCs dendritic cells

NCBI National Center for Biotechnology Information

MHC major histocompatibility complex

CD1 molecules cluster of differentiation 1

T-cells lymphocytes matured in the thymus

IFN-γ interferon-gamma

B-cells lymphocytes matured in the bone marrow

NK-cells natural killer cells

LPS lipopolysaccharide

PRRs pattern recognition receptors

PAMPs pathogen associated molecular pattern

FcγR Fc gamma receptor

NFĸB nuclear factor kappa-light-chain-enhancer of

activated B-cells

LM lipomannan

PIM phosphatidyl mannosides

DNA deoxyribonucleic acid

CpG motifs cytosine phosphate guanine motif

MDP muramyl dipetide

LAM lipoarabinomannan

IgG immunoglobulin G

ITAM cytosolic immunoreceptor-based motif


184

DAG diacylglycerol

ATP adenosine triphosphate

GTP guanine triphosphate

ABP actin binding proteins

NPF nucleation promoting factor

WASP Wiskott-Aldrich syndrome proteins

PI phosphatidylinositol

SNARE soluble N-ethylmaleimide-sensitive factor

attachment protein

TfR transferring receptor

ILVs intraluminal vesicles

LAMP lysosome-associated membrane protein

LBPA lyso-bisphosphatic acid

VAMP vesicle-associated membrane protein

pH potentia hydrogenii

ROI reactive oxygen species

NADPH nicotinamide adenine dinucleotide phosphate

RNI reactive nitrogen species

RNA ribonucleic acid

NO nitric oxide

O2 oxygen

ONOO- peroxinitrite

CO2 carbon dioxide

NO2 nitrogen dioxide

H2O2 hydrogen peroxide

H37Rv M. tuberculosis laboratory reference strain

sec seconds

min minute (s)

h hours (s)

% percent

OMP outer membrane protein

MOM mycobacterial outer membrane

AG arabinogalactan

PG peptidoglycan

PM plasma membrane

http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide_phosphate


185

PG phosphatidylglycerol

PE phosphatidylethanolamine

ManLAM mannosylated lipoarabinomannan

TMM trehalose monomycolate

HPLC high performance liquid chromatography

MS mass spectrometry

v/v volume concentration

MOI multiplicity of infection

BSA bovine serum albumin

RT room temperature

°C degrees Celsius

u units

β beta

M molar

1D-PAGE 1 dimension polyacrylamide gel electrophoresis

V Volt

mm3 cubic milli meter

ON over night

PBS phosphate buffered saline

OD optical density

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel

electrophoresis

PVDF polyvinylidene fluoride

CFU colony forming units

rpm rounds per minute

FT-MS Fourier transformation mass spectrometry

LC liquid chromatography

ESI electron spray ionization

MALDI matrix assisted laser desorption ionization

Qq-TOF-MS quadrupole time of flight mass spectrometry

m/z mass to charge ratio

Da Dalton

FACS fluorescence-activated cell sorting

mpi minutes post infection

hpi hs post infection

http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&ved=0CDwQFjAC&url=http%3A%2F%2Fen.wikipedia.org%2Fwiki%2FPolyvinylidene_fluoride&ei=9hB0VY_XH6TmyQPNr4OADw&usg=AFQjCNG87OFrSaYqrtFPlbYPMf0VxhAKjQ&bvm=bv.95039771,d.bGQ


186

siRNA small interference ribonucleic acid

NTR non-targeting RNA

dpt days post transfection

d deci

c centi

m milli

µ micro

n nano

p pico

f femto

l liter

M molar

g Gramm

Cer ceramides

Cho cholesterol

LBPA lyso-bisphosphatic acid

LPC lyso phosphatidylcholine

PC phosphatidylcholine

PC-O plasmalogen phosphatidylcholine

PE phosphatidylethanolamine

PE-O plasmalogen phosphatidylethanolamine

PG phosphatidylglycerol

PI phosphatidylinositol

PS phosphatidylserine

SM sphingomyeline

PA phosphatidic acid

LBP latex bead phagosomes

PCV pathogen containing vacuole

ER endoplasmic reticulum

EUSC exclusive unique spectrum count

Ca2+ calcium

LatA latrunkulin A

Publications and Conferences

187

Publications and Conferences

Publications

J. Herweg, N. Hansmeier, A. Otto, A.C. Geffken, P. Subbarayal, B. K. Prusty,

D. Becher, M. Hensel, U. E. Schaible, T. Rudel and H. Hilbi, “Purification and

proteomics of pathogen-modified vacuoles and membranes”, in Frontiers in

Microbiology, submitted

AC. Geffken, E. C. Patin, and U. E. Schaible, “Chapter 22 : Isolation of Bead-

Phagosomes Bead Phagosomes to Study Virulence Function of M . tuberculosis

Cell-wall Lipids,” in Mycobacteria Protocols, Third Edit., T. Parish and D. Roberts,

Eds. Springer, 2015, pp. 1–10.

M. Kolonko, A. C. Geffken, T. Blumer, K. Hagens, U. E. Schaible, and M.

Hagedorn, “WASH-driven actin polymerization is required for efficient

mycobacterial phagosome maturation arrest.,” Cell. Microbiol., vol. 16, no.

October 2013, pp. 232–246, Sep. 2013.

Selected conferences

LCI-Symposia, “Emerging Infections”, 29.-30.01.2015, Hamburg

Keystone Symposia “Host Response in Tuberculosis”, 21.-28.01.2015, NM, USA

Poster: M. tuberculosis – Host-Pathogen Interactions in the Phagosome

3rd International Symposia for PhD-Students, “Protein Trafficking in Health and

Disease”, 10.-12.09.2014, Hamburg


Scientific Advisory Board Meeting, 30.09.2014, Borstel

Poster: M. tuberculosis – Wirtszellinteraktionen im Phagosom

1st Poster-Price, 300 € Travel Grand

International Proteomics Summer School, 14.-19.07.2014, Greifswald

LCI-Symposia, “Pathogenesis of Infections”, 30.-31.01.2014, Hamburg

Symposium “Systems Biology of Infections”, 23.06-27.06.2013, Lugano,

Switzerland


17th DGFI-Symposia, “Infektion und immunabwehr“, 21.-23.03.2013, Burg

Rothenfels

Talk: WASH-driven F-actin is required for efficient mycobacterial phagosome

maturation arrest

3rd Presentation Price

Acknowledgements

188

Acknowledgements

First, I would like to thank my supervisor Prof. Dr. Ulrich Emil Schaible for giving me

the opportunity to pursue my PhD studies his lab group “Cellular Microbiology” in

Borstel as well as for his guidance throughout the course of this work.

I would also like to thank my committee members, Prof. Dr. Jan Rupp and Prof. Dr.

Norbert Tautz (University of Lübeck).

Many thanks to Prof. Dr. Dörte Becher and Dr. Andreas Otto (University of Greifswald)

for the excellent cooperation regarding mass spectrometry of my bead phagosome

proteome. You really helped me a lot with my mass spectrometry studies. In addition,

I always had a really nice time during my research visits in Greifswald and during the

Proteomics Summer School.

In addition, many thanks also to Dr. Dominik Schwudke and Dr. Nicole Zehethofer for

the excellent cooperation regarding the mass spectrometry of my phagosome

lipidome. I really enjoyed spending time at the Parkallee 10 eating a lot of your sweets

A big thank you also to Prof. Dr. Thomas Gutsmann for being my co-supervisor and

all the fruitful discussions.

I would also like to thank Kristine Hagens, Jacqueline Eich, Nina Grohmann and

Dagmar Meyer for your great technical assistance in the lab. Further, I would like to

thank Martina Hein, Dr. Jochen Behrends and Dr. Thomas Scholzen for technical

assistance and a lot of patience with the confocal microscope and the FACS machines.

Further, I would like to thank the Cellular Microbiology Group, present and former

members: Maike Burmeister, Carlotta Ober-Blöbaum, Jannike Blank, Dr. Tobias

Dallenga, Bhesh Paudyal, Lars Eggers, Dr. Natalja Redinger, Dr. Bianca Schneider,

Dr. Yeojun Yun, Dr. Matthias Hauptmann, Dr. Steffi Renk for the great working

environment. Working and going to conferences with you guys was a lot of fun all the

time Moreover, I would like to thank “my” trainee Christoph Leschczyk for being a

great help during the last 8 month! I wish you all the best for your future and of course

a PhD-project without Western blots

Furthermore, I would like to thank my family. Papa Helmut, Mama Gesa, Bruder Yves,

Oma Irmgard (2x), Opa Heinz und Walter, Onkel Peter und Cousin Lucas. Thank you

a lot for your patience with me in the last 6 month Kim and Denise, thanks for your

friendship

Robert, this is for you ♥ !

189

Erklärung

Hiermit versichere ich an Eides statt, dass ich die vorliegende Dissertation

selbstständig angefertigt habe und keine anderen Hilfsmittel als angegeben verwendet

habe. Weder vor noch gleichzeitig habe ich andernorts einen Zulassungsantrag

gestellt oder diese Dissertation vorgelegt. Ich habe mich bisher noch keinem

Promotionsverfahren unterzogen. Ferner bin ich damit einverstanden, dass die

vorliegende Dissertation von der Frankfurter Bibliothek gespeichert und im Internet

veröffentlicht wird.

Hamburg, den

__________________________

Anna Christina Geffken

mycobacterium tuberculosis - host-cell interactions in the

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