ANTIBIOTIC UPTAKE INGRAM-NEGATIVE BACTERIA

Claudio Muheim

Antibiotic uptake inGram-negative bacteria

Claudio Muheim

©Claudio Muheim, Stockholm University 2017 ISBN print 978-91-7797-041-5ISBN PDF 978-91-7797-042-2 Printed in Sweden by Universitetsservice US-AB, Stockholm 2017Distributor: Department of Biochemistry and Biophysics, Stockholm University

To the decentralized future.

List of publications

I. YfgM is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli Götzke H, Palombo I, Muheim C, Perrody E, Genevaux P, Kudva R, Müller M, Daley DO. J Biol Chem (2014) 289(27), 19089-19097

II. Identification of Putative Substrates for the Periplasmic

Chaperone YfgM in Escherichia coli Using Quantitative Proteomics Götzke H, Muheim C, Maarten AF, Heck AJ, Maddalo G, Daley DO. Mol Cell Proteomics (2015) 14(1), 216–226

III. Increasing the Permeability of Escherichia coli using

MAC-13243 Muheim C, Götzke H, Eriksson AU, Lindberg S, Lauritsen I, Nørholm MHH, Daley DO. Submitted

IV. Identification of a Fragment-Based Scaffold that Inhibits the

Glycosyltransferase WaaG from Escherichia coli. Muheim C, Bakali A, Engström O, Wieslander Å, Daley DO, Widmalm G.

Antibiotics (2016) 5(1), 10

Author’s contribution to the publications

I. I performed the antibiotic disc diffusion assays and was involved in the interpretation of the results.

II. I cloned the plasmid constructs and performed the acid-stress

assays.

III. I was involved in the design of all experiments. I performed all of the experiments apart from the high-throughput screen. I contributed significantly in writing the final manuscript.

IV. I designed and performed all experiments apart from the in vitro lipid binding assay and the O-Deacylation of LPS. I contributed significantly in writing the final manuscript.

Abstract

The increasing emergence and spread of antibiotic-resistant bacteria is a

serious threat to public health. Of particular concern are Gram-negative

bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella

pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant

to a large number of antibiotics and thus our treatment options are rapidly

declining. In addition to the increasing number of antibiotic-resistant

bacteria, a major problem is that many of the antibiotics at our disposal are

ineffective against Gram-negative bacteria. This is partly due to the

properties of the outer membrane (OM) which prevents efficient uptake. The

overarching goal of this thesis was to investigate how the OM of the Gram-

negative bacterium E. coli could be weakened to improve the activity of

antibiotics.

In the first two papers of my thesis (paper I + II), I investigated the

periplasmic chaperone network which consists of the two parallel pathways

SurA and Skp/DegP. This network is essential for the integrity of the OM

and strains lacking either SurA or Skp are defective in the assembly of the

OM, which results in an increased sensitivity towards vancomycin and other

antimicrobials. We identified a novel component of the periplasmic

chaperone network, namely YfgM, and showed that it operates in the same

network as Skp and SurA/DegP. In particular, we demonstrated that deletion

of YfgM in strains with either a surA or skp background further

compromised the integrity of the OM, as evidenced by an increased

sensitivity towards vancomycin.

In the remaining two papers of my thesis (paper III + IV), the goal was to

characterize small molecules that permeabilize the OM and thus could be

used to improve the activity of antibiotics. Towards this goal, we performed

a high-throughput screen and identified an inhibitor of the periplasmic

chaperone LolA, namely MAC-13243, and showed that it can be used to

permeabilize the OM of E. coli (paper III). We further demonstrated that

MAC-13243 can be used to potentiate the activity of antibiotics which are

normally ineffective against E. coli. In the last paper of my thesis (paper IV),

we undertook a more specific approach and wanted to identify an inhibitor

against the glycosyltransferase WaaG. This enzyme is involved in the

synthesis of LPS and genetic inactivation of WaaG results in a defect in the

OM, which leads to an increased sensitivity to various antibiotics. In this

paper, we identified a small molecular fragment (compound L1) and showed

that it can be used to inhibit the activity of WaaG in vitro.

To summarize, this thesis provides novel insights into how the OM of the

Gram-negative bacterium E. coli can be weakened by using small molecules.

We believe that the two identified small molecules represent important first

steps towards the design of more potent inhibitors that could be used in

clinics to enhance the activity of antibiotics.

Contents

List of publications .................................................................................... viii

Author’s contribution to the publications ................................................. ix

Abstract .......................................................................................................... x

Contents ...................................................................................................... xii

Abbreviations ............................................................................................. xiv

Introduction ................................................................................................. 16

1.1 Antibiotics .......................................................................................... 16

1.2 Antibiotic resistance ........................................................................... 18

1.3 The antibiotic crisis ............................................................................ 20

1.3.1 The lack of novel antimicrobials ................................................ 21

1.3.2 The excessive use and misuse of antibiotics ............................... 22

1.3.3 The lack of infection control and surveillance programs ............ 23

1.4 The emerging threat of Gram-negative bacteria ................................. 24

1.5 Structural overview of the Gram-negative cell envelope ................... 25

1.5.1 The outer membrane ................................................................... 26

1.5.2 The periplasmic space ................................................................. 28

1.5.3 The inner membrane ................................................................... 28

1.6 Antibiotic transport across the Gram-negative cell envelope ............. 29

1.6.1 Porin-mediated antibiotic uptake ................................................ 29

1.6.2 Diffusion across the LPS ............................................................ 30

1.6.3 Diffusion across the periplasmic space and IM .......................... 31

1.6.4 Efflux pumps .............................................................................. 33

1.7 Antibiotic adjuvants ........................................................................... 34

1.8 Additional targets for antibiotic adjuvants ......................................... 37

Summary of the papers .............................................................................. 44

Conclusions and future perspectives ......................................................... 57

Populärvetenskaplig sammanfattning....................................................... 61

Acknowledgements ..................................................................................... 62

References .................................................................................................... 64

Abbreviations

A. baumannii Acinetobacter baumannii

ABC ATP-binding cassette

BAM -barrel assembly machinery

CL Cardiolipin

E. coli Escherichia coli

EPIs Efflux pump inhibitors

HEP L-glycero-D-manno-heptose

IM Inner membrane

KDO 3-deoxy-D-manno-oct-2-ulosonic acid

K. pneumoniae Klebsiella pneumoniae

LPS Lipopolysaccharide

MATE Multidrug and toxic compound extrusion

MIC Minimal Inhibitory Concentration

MDR Multidrug resistance

MFS Major facilitator superfamily

NPN N-phenyl-1-naphthylamine

OM Outer membrane

OMP Outer membrane protein

OS Oligosaccharide

PA N Phenylalanine-arginine -naphthylamide

PE Phosphatidylethanolamine

PG Phosphatidylglycerol

P. aeruginosa Pseudomonas aeruginosa

PMBN Polymyxin B nonapeptide

RND Resistance-nodulation-division

SMR Small multidrug resistance

TPR Tetratricopeptide repeat

WT Wild type

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Introduction

1.1 Antibiotics

Microbes, such as bacteria and fungi synthesize antibiotics and secrete them

into the environment to inhibit growth of competing microbes. Millions of

years of chemical warfare between rivalling species formed an arsenal of

antibiotics that we use today to treat bacterial infections (1, 2). Based on

their antimicrobial activity, antibiotics can be broadly classified into two

groups: bacteriostatic antibiotics arrest the growth of bacteria but do not kill

them whereas bactericidal antibiotics induce cell death (Figure 1). However,

it is often difficult to classify an antibiotic into one of these categories since

antibiotics can have both bacteriostatic and bactericidal activity. Various

factors such as growth conditions, bacterial density, antibiotic concentration

or antibiotic exposure time influence the antimicrobial activity (3, 4). Thus,

antibiotics are also categorized into different classes, based on their

structure, mechanism or spectrum of activity. One of the most prominent

classifications is based on the chemical structure, since antibiotics with

structural similarities typically show similar effects regarding their activity

and toxicity.

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Figure 1. Antimicrobial effect of bacteriostatic and bactericidal antibiotics.

Based on their antimicrobial effect, antibiotics can either inhibit bacterial growth

(green curve) or induce cell death (red curve). Various factors such as growth

conditions, bacterial density, antibiotic concentration or antibiotic exposure time

influence the antimicrobial activity. Figure adapted from (5). Reprinted with

permission.

Despite approximately 3,000 known antibiotics, most of their cellular targets

have not been characterized yet (6). Interestingly, those antibiotic targets

which have been identified can be assigned to a few cellular processes (7).

These include cellular processes which are essential for the survival of

bacteria such as DNA replication, RNA transcription, protein translation,

cell wall synthesis, the cell membrane and a few other metabolic pathways

(Figure 2).

One of the most successful antibiotic classes are the -lactams. -lactams are

a class of broad-spectrum antibiotics which inhibit cell wall synthesis (8). In

particular, -lactams prevent crosslinking of the peptidoglycan units by

inhibiting peptide bond formation which is catalysed by penicillin-binding

proteins. Many different classes of -lactam antibiotics have been introduced

into the clinics which include the penicillins, the cephalosporins, the

cephamycins, the carbapenems and the monobactams. However, despite

their unarguable success, -lactams and other antibiotics have been

18

challenged by the problem of increasing bacterial resistance. As a

consequence, these antibiotics need to be constantly evolved in order to

evade bacterial resistance.

Figure 2. Antibiotic classes inhibit various cellular processes. Most antibiotic classes

inhibit only a few cellular processes. These are often conserved among bacteria and

include DNA replication, transcription, translation, cell wall synthesis, the cell membrane

and a few other metabolic pathways (not shown).

1.2 Antibiotic resistance

In the traditional sense, antibiotic resistance occurs when a bacterial species

acquires the ability to resist the action of an antibiotic. This process is also

known as acquired resistance and can either happen by mutation of the

bacterial chromosome or by horizontal gene transfer of foreign DNA coding

for antibiotic resistance elements. Horizontal gene transfer is particularly

problematic since it largely contributes to the spread of antibiotic resistance

elements within and even across bacterial species (9). On the other hand,

bacterial species can be naturally resistant to certain antibiotics. These

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species have the ability to withstand the action of an antibiotic due to its

inherent structural or functional properties. These properties are encoded in

the bacterial genome and ensure that bacteria are intrinsically resistant to

certain antibiotics.

The general mechanisms which confer resistance to antibiotics are depicted

in Figure 3. These include (1) utilizing an alternative metabolic pathway

which bypasses the inhibited one, (2) modification of the antibiotic target

that prevents or reduces binding of the antibiotic, (3) overproduction of the

antibiotic target, (4) enzymatic inactivation or modification of the antibiotic,

(5) active efflux of the antibiotic by efflux pumps and (6) reduced antibiotic

uptake by a decreased permeability of the outer membrane (OM) (10).

Figure 3. Antibiotic resistance mechanisms. Bacterial cells have general mechanisms to

resist the action of an antibiotic. These include (1) bypassing the inhibited pathway, (2)

alteration of the target, (3) amplification of the target, (4) enzymatic inactivation or

modification of the antibiotic, (5) increased antibiotic efflux and (6) reduced antibiotic

uptake. Figure adapted from (10). Reprinted with permission.

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1.3 The antibiotic crisis

The discovery of the -lactam antibiotic penicillin by the Scottish scientist

Alexander Fleming in 1928 is considered as a key event in modern medicine

(11). After being introduced into the clinics in the 1940s, penicillin saved

thousands of lives. For example, it was used to treat pneumococcal wound

infections that were caused by battlefield injuries, and as a consequence, the

survival rate of injured soldiers significantly improved (12). The great

success of penicillin initiated a golden rush in antibiotic drug discovery.

More than 20 new antibiotic classes were discovered from 1940 to 1962

(Figure 4) (13).

Figure 4. Timeline of antibiotic discovery. Figure adapted from (14).

Reprinted with permission.

Notably, most antibiotics which are currently in clinical use were discovered

during this period. However, despite ongoing efforts, this productive phase

abruptly ended in the 1960s and since then only two new antibiotic classes

21

were clinically approved for human use, namely the oxazolidinones in 2000

and the lipopeptides in 2003 (15). These two classes were discovered

decades ago (Figure 4) and illustrate how long it can take until a novel

antibiotic class enters into the market. It is estimated that we need a further

20 novel antibiotic classes to support modern medicine during the next 50

years (13).

There are several reasons which are believed to be the driving forces of the

antibiotic crisis. As previously described, the number of novel antibiotic

classes entering into the market has been steadily declining over the last few

decades. In addition, the number of multidrug resistant bacteria has been

increasing due to an excessive use of antibiotics. Finally, the global spread

of multidrug resistant bacteria has been increasing due to an enhanced global

connectivity. In the next few sections, I will discuss each of these points and

explain some of the major problems.

1.3.1 The lack of novel antimicrobials

The antibiotic pipeline is drying out and the situation is deteriorating (16).

After the antibiotic gold rush ended in the 1960s, pharmaceutical companies

shifted their focus on analogue development because of a gradually

declining number of novel antibiotic classes. The toxicity risks of analogues

were considered to be more predictable compared to new antibiotic classes

(13). Despite the risk of cross-resistance, analogue development yielded a

number of new antibiotics that kept up with the emergence of bacterial

resistance until the last two decades (13). Since then, however, many

pharmaceutical companies have completely abandoned antibiotic drug

discovery and development programs. It is no longer considered to be an

attractive investment because of regulatory obstacles, increasingly restricted

22

antibiotic use and uncertainties about emerging resistance (17–19).

Additional efforts to develop new platforms for antibiotic drug discovery,

such as high-throughput screening against defined targets or rational drug

design were largely unsuccessful (6, 20). Although there is increasing

evidence that pharmaceutical companies are restarting antibiotic drug

discovery programs, it remains unclear if the antibiotic pipeline can be filled

to its current need (21).

1.3.2 The excessive use and misuse of antibiotics

Antibiotics are among the most commonly purchased drugs (22). There is

clear evidence that antibiotic resistance is a direct consequence of excessive

antibiotic use (23–25). It is estimated that about half of all antibiotics

prescribed to patients are not required or inadequately prescribed (26).

Antibiotic misuse is especially problematic in countries which have no

regulations regarding antibiotic dispensation. In these countries, antibiotics

are sold without prescription and dispensed by people lacking medical

knowledge (27). Another major problem is the use of antibiotics as growth

supplements in livestock (28, 29). In this common practice, a sub-therapeutic

concentration of antibiotic is mixed to the feed to improve the feed

efficiency. Some of these growth promotors belong to antibiotic classes

which are used in human medicine and it has been reported that this can lead

to cross-resistance (30). Another and often neglected problem is the

environmental contamination with antibiotics by pharmaceutical production

facilities in developing countries. Antibiotic waste products end up

unfiltered in natural waters, which causes a selection pressure on bacteria

that eventually leads to the emergence of antibiotic-resistant bacteria (31).

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1.3.3 The lack of infection control and surveillance

programs

Antibiotic resistance is an international problem and the increase in global

connectivity is partly responsible for the transmission of multidrug resistant

bacteria across borders. Multidrug-resistant bacteria often end up in

healthcare facilities where they spread among patients and healthcare

professionals (32). In addition, there is often a lack of infection control and

surveillance programs in these facilities which further promotes the

transmission of drug-resistant bacteria (33). As a consequence, thousands of

people die every year from hospital-acquired infections (34). Hence, various

antimicrobial stewardship programs have been initiated to address this issue

(35–37). These programs are coordinated at a national and international level

and address points to limit transmission rates within healthcare facilities and

community settings as well as monitoring antibiotic resistance and antibiotic

use.

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1.4 The emerging threat of Gram-negative bacteria

It is evident that we are entering into a period with a lack of novel antibiotics

to treat bacterial infections. The discovery of novel antibiotics is not keeping

up with the emergence of antibiotic resistant bacteria. Although resistance to

antibiotics is a general problem, the increasing number of infections caused

by multidrug resistant Gram-negative bacteria is alarming (38, 39). Highly

resistant pathogens such as Escherichia coli, Acinetobacter baumannii,

Klebsiella pneumoniae or Pseudomonas aeruginosa are among the most

critical threats (26, 40). These pathogens are of particular concern in both

healthcare facilities and community settings (41). It is no longer uncommon

that some of these strains are resistant to nearly all available antibiotics.

Thus, the number of treatment options rapidly decline and clinicians are

forced to use older drugs, such as the last-resort antibiotic colistin, which has

been associated with neuro- and nephrotoxicity (42). The situation is

alarming and we need new strategies to tackle the problem of Gram-negative

resistance. It is worth mentioning that the unique architecture of the Gram-

negative cell envelope is partly responsible for the problem. A combination

of limited antibiotic uptake across the OM and efflux of antibiotics by

multidrug efflux pumps limit the activity of many antimicrobials. Since the

work covered in this doctoral thesis has aimed to focus on the antibiotic

uptake problem, the following sections will introduce a structural overview

of the Gram-negative cell envelope whilst describing the properties that

make the cell envelope largely impermeable to most antibiotics.

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1.5 Structural overview of the Gram-negative cell

envelope

The cell envelope is a complex structure which is involved in various

cellular processes, such as protection of the cell and maintenance of cellular

shape (43). It is composed of three essential layers, namely an OM that faces

towards the exterior, a periplasmic space that includes a thin layer of

peptidoglycan and an inner membrane (IM) that separates the periplasm

from the cytosol (Figure 5).

Figure 5. The cell envelope of Gram-negative bacteria. The Gram-negative cell

envelope consists of an OM that provides a barrier to the extracellular environment, a

periplasmic space which includes a thin layer of peptidoglycan and an IM that separates

the periplasmic space from the cytosol. The OM contains lipopolysaccharides (LPS) and

various outer membrane proteins (OMPs), which have a characteristic -barrel structure.

The IM contains integral membrane proteins as well as peripherally attached membrane

proteins. In addition, both membranes contain various lipoproteins which face towards the

periplasmic side. Adapted from (44). Reprinted with permission.

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1.5.1 The outer membrane

The OM is the outermost barrier, which separates the extracellular

environment from the inner compartment of the bacterial cell. It is an

asymmetric lipid bilayer that consists of two leaflets: the inner leaflet

predominantly contains phospholipids, in particular

phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), as well as a

small fraction of cardiolipin (CL) (45). The outer leaflet faces towards the

exterior and is mainly composed of the glycolipid lipopolysaccharide (LPS)

(46). LPS consists of three separate regions: a hydrophobic lipid A tail that

anchors the LPS molecule to the OM, a phosphorylated core oligosaccharide

(OS) and a repeating OS unit which is known as the O-antigen

polysaccharide (Figure 6).

Figure 6. Structural composition of LPS from E. coli K12. The LPS molecule can be

structurally divided into three parts: a lipid A part which embeds the LPS molecule into

the OM, a core OS which consists of a conserved inner core and a less conserved outer

core, and an O-antigen which is the outermost part of the LPS molecule. LPS is a

heterogeneous molecule and can have various modifications in each part (47).

Abbreviations: Hep, L-glycero-D-manno-heptose; Glu, glucose; Gal, galactose; P,

phosphate; PEtN, phosphoethanolamine; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid;

GlcN, glucosamine. Adapted from (48). Reprinted with permission.

27

The lipid A part (also known as endotoxin) is highly hydrophobic and

activates the adaptive immune response (49). It consists of a phosphorylated

diglucosamine backbone with covalently linked acyl chains that anchor the

LPS molecule into the OM. The structure of lipid A varies among bacterial

species and modification of the lipid A component can affect the endotoxic

activity (47). For example, lipid A modifying enzymes affect the number,

position and length of acyl chains as well as the phosphorylation pattern or

the sugar composition of the disaccharide backbone (50). The core OS is

attached to the lipid A and divided into two parts: the inner core is largely

conserved in Gram-negative bacteria and composed of the sugars 3-deoxy-D-

manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep).

The outer core is less conserved and composed of different hexose sugars.

Both the inner and outer core can be modified by the addition of phosphates,

pyrophosphates, various phospholipids and amino acids (47). The outermost

part of the LPS molecule is the O-antigen which is linked to the core OS.

The O-antigen is the major antigen targeted by the host immune response

and consists of repeating units of oligosaccharides with high variability (51).

Each of these repeating units contains one to eight glycosyl residues and

varies between sequence, chemical linkage and sugar composition. The OM

is also spanned by outer membrane proteins (OMPs). OMPs have a

characteristic -barrel structure and mediate transport, signalling and other

vital functions (52). Some of these OMPs form water-filled channels which

are also known as porins. Porins regulate the uptake of nutrients, ions and

other molecules such as antibiotics (53, 54). The OM also contains

lipoproteins, which are anchored through an N-terminal lipid moiety to the

inner leaflet of the OM. In E. coli, there are ~ 100 OM lipoproteins which

play important roles in the assembly of the OM (55). The most abundant

lipoprotein in E. coli is Lpp with more than 500,000 copies per cell (43). Lpp

provides structural stability to the cell envelope by covalently linking the

OM with the underlying peptidoglycan layer (Figure 5) (56, 57).

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1.5.2 The periplasmic space

The periplasmic space is an aqueous, protein-filled environment that lies

between the IM and OM (Figure 5). This compartment contains various

proteins and polysaccharides that regulate cellular processes such as nutrient

uptake, detoxification of harmful compounds or protein transport and quality

control (58). It also includes a thin layer of peptidoglycan that stabilizes the

cell membranes against internal osmotic pressures (43). The peptidoglycan

layer is composed of linear glycan strands that consist of alternating units of

N-acetylglucosamine and N-acetylmuramic acid, which are connected by -

(1,4)-glycosidic bonds (59). The glycan strands are crosslinked by short

peptide linkages which create a net-like polymeric structure.

1.5.3 The inner membrane

The IM separates the periplasm from the cytosol and is composed of two

leaflets that form a symmetric phospholipid bilayer. The phospholipid

composition of the IM is comparable to the inner leaflet of the OM. In E.

coli, the IM consists of 70 – 80 % PE, 15 – 20 % PG and a small fraction of

CL (60, 61). The IM is composed of various proteins that mediate essential

cellular functions such as selective transport, energy metabolism, cell

division, motility, and signalling (62). IM proteins are generally divided into

two classes: integral membrane proteins contain one or more -helical

transmembrane segments that span the phospholipid bilayer. Peripherally

attached membrane proteins do not span the IM but instead adhere to one of

the leaflets via electrostatic, hydrophobic or non-covalent interactions.

Another group of proteins which interacts with the IM are lipoproteins.

Lipoproteins are anchored to the outer leaflet of the IM through their N-

terminal lipid moieties and involved in various processes of the bacterial cell

envelope (63).

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1.6 Antibiotic transport across the Gram-negative

cell envelope

All antibiotics need to cross one or both membranes of the cell envelope in

order to reach their intracellular target. In addition to the physical barriers

provided by the bacterial membranes, multiple efflux pumps reduce the

intracellular antibiotic concentration. The first and most relevant barrier that

antibiotics encounter is provided by the OM (53, 64). Antibiotics have two

choices to cross this OM: they can either diffuse through water-filled porin

channels or permeate through the LPS-containing leaflet of the OM. In this

section, I will address how the various barriers of the cell envelope limit the

efficient uptake of certain antibiotics in Gram-negative bacteria.

1.6.1 Porin-mediated antibiotic uptake

The uptake of hydrophilic antibiotics < 600 Daltons (herein termed as small-

scaffold antibiotics) is largely regulated by water-filled channels known as

porins (Figure 7) (53, 54, 65). Porins can be classified into different groups

according to their structure, regulation, expression and activity (66). General

diffusion porins form trimers of 16-stranded -barrels and allow the passage

of hydrophilic molecules with limited substrate selectivity (54, 64). In

contrast, substrate-specific porins form trimers of 18-stranded -barrels and

are selective for specific substrate classes such as higher oligosaccharides

(53). Most porins which are involved in antibiotic transport belong either to

the OmpC or OmpF subfamilies (54). These porins represent the main entry

pathway for small, hydrophilic antibiotics such as -lactams or

fluoroquinolones (54, 67). E. coli has three major diffusion porins, namely

OmpC, OmpF and PhoE. OmpC and OmpF porins have a slight preference

30

for cations whereas PhoE prefers inorganic phosphate and anions (53, 65).

Other factors such as molecular shape, number of rotatable bonds and the

presence of an ionisable nitrogen group also affect diffusion across porin

channels (68).

Bacterial cells have different strategies to acquire resistance to antibiotics

which diffuse through porin channels. This can be caused by exchanges in

the porin type, changes in the expression profile of porins or by mutations or

modifications which affect the channel properties of porins (54). For

example, a study showed that clinical isolates of K. pneumonia had a

reduced OM permeability after antibiotic treatment due to a change in the

expression of porins from large channel size OmpF to the smaller channel

size OmpC (69). In addition, some Gram-negative bacteria such as P.

aeruginosa possess a high intrinsic resistance towards many antibiotics (e.g.

-lactams) which is partly caused by the low number of general diffusion

porins (53).

1.6.2 Diffusion across the LPS

Antibiotics larger than 600 Daltons (herein called large-scaffold antibiotics)

do not efficiently cross porin channels (Figure 7). They can permeate

through the LPS-containing OM but their diffusion is often limited. It has

been shown that lipophilic molecules permeate much less efficiently across

the OM bilayer than through a standard phospholipid bilayer (70, 71). In

addition, the LPS-containing bilayer largely restricts the diffusion of

hydrophilic molecules. Hence, the OM is an effective barrier against both

hydrophobic as well as hydrophilic molecules (53, 72).

The impermeability of the OM can be mainly attributed to the LPS layer.

This layer is stabilized by divalent cations, such as Mg2+ and Ca2+, which

31

cross-bridge negatively charged LPS molecules and thus stabilize the lateral

interaction between neighbouring LPS molecules (53). Displacement of the

divalent cations by the chelator EDTA destabilizes the LPS layer (73).

Subsequently, LPS is released into the medium and it is assumed that

phospholipids from the inner leaflet compensate for the loss. As a

consequence, Gram-negative bacteria become sensitized towards

hydrophobic antibiotics including erythromycin, rifampicin and novobiocin

(74). The core region of LPS plays an important role and contributes to the

barrier function (64). Strains expressing full-length LPS (termed smooth

LPS) are intrinsically resistant to hydrophobic and large-scaffold antibiotics.

However, strains expressing truncated LPS (termed rough or deep rough

LPS) are more susceptible (64, 75). Deep rough mutants which have the

most truncated core are also highly susceptible to lipophilic agents such as

detergents, bile salts and some antibiotics (64).

1.6.3 Diffusion across the periplasmic space and IM

Antibiotics that have crossed the OM arrive in the periplasmic space. This

compartment is the site of action for antibiotics such as the -lactams or the

glycopeptide antibiotic vancomycin. Little is known about the diffusion of

antibiotics through this compartment but it is believed that the periplasm

represents no major barrier for antibiotics (76). Those antibiotics destined for

the cytosol need to penetrate the IM. The phospholipid bilayer of the IM is

largely permeable to lipophilic antibiotics (67, 77). Thus, relatively

hydrophobic antibiotics such as the macrolides, lincosamides,

oxazolidinones, pleuromutilins streptogramins A and B, the elfamycins, and

rifamycins appear to cross the IM by simple diffusion (78). In contrast, the

IM is largely impermeable to large, uncharged polar molecules and all

32

charged molecules including ions (79). Those antibiotics which belong to

these categories need to cross the IM by specific uptake systems. For

example, D-cycloserine is transported across the IM via the D-alanine

transport system and coupled to the proton motive force (80). Another

example is fosfomycin which is transported across the IM by using the

glycerol-3-phosphate or the hexose phosphate transporters (81). Transport of

aminoglycosides requires both the electrochemical gradient across the IM

and the electron flow through the respiratory chain (82). Despite the

characterization of a few transport systems, antibiotic transport across the IM

remains poorly characterized. Notably, it has also been suggested that all

hydrophilic antibiotics traverse the IM by solute transport systems (83).

Figure 7. Antibiotic uptake in Gram-negative bacteria. Small-scaffold antibiotics (<

600 Da) generally permeate the OM by passively diffusing through non-specific OMPs.

In contrast, large-scaffold antibiotics (> 600 Da) diffuse through the LPS-containing OM

to gain access to the cell interior. However, their diffusion is limited and thus their uptake

inefficient. Both large and small scaffold antibiotics can either diffuse across the IM or

they can be inadvertently taken up by membrane-embedded transporters.

33

1.6.4 Efflux pumps

Efflux pumps are conserved in nearly all bacterial species and the genes

encoding for efflux pumps are either located on the bacterial chromosome or

on transmissible elements such as plasmids (84). Bacterial efflux pumps can

be classified into several families based on the number of components, the

number of transmembrane-spanning regions and the energy source they use

to transport their substrate (85). Efflux pumps can be specific for a substrate

or they can expel a broad range of structurally diverse compounds (86).

There are five families of efflux pumps which are associated with multidrug

resistance (MDR): these include the ATP-binding cassette (ABC)

superfamiliy, the major facilitator superfamily (MFS), the multidrug and

toxic compound extrusion (MATE) family, the small multidrug resistance

(SMR) family and the resistance-nodulation-division (RND) family (85).

The ABC family transporters use the energy of ATP-hydrolysis for drug

export whereas the others are antiporters and dependent on the H+ (or Na+)

proton gradient (87). The major relevant efflux pumps in Gram-negative

bacteria belong to the RND-type family which is often associated with

multidrug resistance in clinical isolates (67). Efflux pumps of the RND-type

family are organised as tripartite systems which are composed of an IM

transporter, a periplasmic adapter protein and an OM protein. RND-type

efflux systems are polyspecific and expel a broad range of substrates (88).

Two of the best studied RND-type efflux pumps in Gram-negative bacteria

are the AcrAB-TolC system from E. coli and the MexAB-OprM system

from P. aeruginosa. The AcrAB-TolC efflux pump from E. coli is

constitutively expressed and has a broad substrate profile which includes

antimicrobials such as fluoroquinolones, lipophilic -lactams,

chloramphenicol, rifampicin, novobiocin, tetracycline and fusidic acid (85,

89, 90). Several Acr efflux pumps have been chraracterized in E. coli (91–

93). However, AcrAB-TolC has been found to be the most relevant one in

clinical isolates (94–96). In P. aeruginosa, several RND-type efflux pumps

34

have been characterized among which MexAB-OprM is the major multidrug

efflux system (97). MexAB-OprM is consitutively expressed in wild type

(WT) P. aeruginosa and involved in the transport of various antimicrobials

such as fluoroquinolones, -lactams, macrolides, tetracyclines, trimethoprim,

sulfamides and chloramphenicol (98). MexAB-OprM contributes

significantly to the high intrinsic resistance and overexpression of it has been

associated with MDR in clinical isolates of P. aeruginosa (99).

1.7 Antibiotic adjuvants

As previously stated, the development of novel antibiotics is not sufficient to

keep up with the increasing number of antibiotic resistant bacteria. Thus,

there is a pressing need to explore alternative strategies to tackle the issue.

One alternative strategy which has received increased attention over the past

few years is the development of adjuvants to potentiate the activity of

existing antibiotics (100–102). Antibiotic adjuvants are compounds which

normally have little or no intrinsic antimicrobial activity. However, when

used in combination with an antibiotic, adjuvants enhance the activity of the

antibiotic. Antibiotic adjuvants can have different cellular targets but they

act by either reversing acquired resistance or sensitizing strains that are

intrinsically resistant (100). Most adjuvants that have been identified

potentiate antibiotics by either inhibiting antibiotic resistance elements (e.g.

inactivation of -lactamases), blocking bacterial efflux pumps, increasing the

permeability of the bacterial cell envelope, or by interfering with signal

systems that are involved in antibiotic resistance (102). In this section, I

would like to focus on a few antibiotic adjuvants which belong to one of

these categories and have been shown to potentiate antibiotics in Gram-

negative bacteria.

35

-lactamase inhibitors are clinically used to improve the activity of -lactam

antibiotics. One of the most successful drugs is Augmentin© which is a

combination of clavulanic acid (a -lactamase inhibitor) and amoxicillin (a

-lactam antibiotic). Augmentin© is used to restore or extend the

antimicrobial activity of amoxicillin in -lactamase-producing strains such

as E. coli and K. pneumoniae (103). Despite the clinical use of Augmentin©

for more than 30 years, the emergence of resistance in clinical isolates has

been very low (104). Potentiation of antibiotics by inactivation of -

lactamases has been of ongoing interest and a few additional drugs are

currently in development (105).

Efflux Pumps are another target to potentiate the activity of antibiotics.

Since most antibiotics are susceptible to active efflux, the use of efflux pump

inhibitors (EPIs) could make antibiotics more effective by increasing their

intracellular concentration. A number of EPIs have been identified in Gram-

negative bacteria (106, 107). One prominent example is the peptidomimetic

compound phenylalanine-arginine -naphthylamide (PA N), which inhibits

the Mex efflux pumps in P. aeruginosa and the homolog AcrAB-TolC in E.

coli (108). PA N potentiated the activity of the fluoroquinolone antibiotic

levofloxacin in P. aeruginosa (108). In particular, PA N reduced the MIC

(Minimal Inhibitory Concentration) of levofloxacin about 8-fold in WT

strains and up to 64-fold in strains overexpressing efflux pumps. PA N also

decreased the MICs of various antibiotics including chloramphenicol,

nalidixic acid, ofloxaxin, cloxaxillin and erythromycin in K. pneumonia

strains that are either naturally sensitive or inherently resistant against these

antibiotics (109). However, despite its good in vitro activity against

clinically relevant multidrug efflux pumps, PA N and its derivatives did not

enter into clinics due to acute toxicity in pre-clinical trials (110).

Another class of antibiotic adjuvants target the bacterial cell envelope. For

example, the OM permeabilizer polymyxin B nonapeptide (PMBN) has been

shown to potentiate various antibiotics in Gram-negative bacteria (74).

36

PMBN improved the activity of erythromycin and novobiocin in mice

infected with either K. pneumonia or P. aeruginosa (111). A combination of

PMBN with either avibactam, cefazidime or cefazidime-avibactam increased

the antimicrobial activity against clinical isolates of E. coli, K. pneumoniae

and E. aerogenes (112). However, despite good in vitro activity against

Gram-negative bacteria, PMBN has been shown to cause a similar toxicity

profile as polymyxin B (113).

Of recent interest are the findings of Wright and colleagues (114): they

screened a collection of previously approved drugs for potentiators of the

tetracycline antibiotic minocycline against E. coli, P. aeruginosa and S.

aureus. The authors identified several non-antibiotic compounds which had

antibiotic adjuvant properties. For example, the anti-diarrheal medication

loperamide improved the activity of minocycline and other tetracycline

antibiotics in either E. coli or P. aeruginosa. Loperamide dissipates the

electrical component of the proton motive force across the bacterial

membrane of Gram-negative bacteria. As a consequence, loperamide

increases antibiotic influx of tetracycline antibiotics. The combination of

loperamide and minocycline was shown to be highly synergistic in a

Salmonella in vivo model. The same group performed an additional screen to

identify antibiotic adjuvants of the aminocoumarin antibiotic novobiocin,

which is normally ineffective against Gram-negative bacteria (115). They

identified four compounds that were shown to be synergistic with

novobiocin in either E. coli or P. aeruginosa. Two of these compounds,

namely A22 and pivmecillinam alter bacterial cell shape by blocking the

cytoskeleton protein MreB and inhibiting peptidoglycan synthesis,

respectively.

37

1.8 Additional targets for antibiotic adjuvants

The number of antibiotic adjuvants is most likely not sufficient to solve the

antibiotic crisis. However, recent genetic studies have provided novel targets

for antibiotic adjuvants by identifying a large number of genes that

contribute to the intrinsic resistance of Gram-negative bacteria. For example,

a transposon mutant library has been used to identify genes that are

responsible for intrinsic resistance to various antibiotics in Acinetobacter

baylyi (116). Other studies have used transposon mutant libraries of P.

aeruginosa to screen for enhanced sensitivity against tobramycin or

ciprofloxacin (117, 118). In addition, Fajardo et al. screened two different

transposon-tagged insertion libraries of P. aeruginosa for increased

susceptibility to six antimicrobials belonging to different structural families

(119). Similar studies have also been done in E. coli. For example, Tamae et

al. and Liu et al. screened an E. coli knockout collection to look for mutants

which are more susceptible to various antibiotics (75, 120). Taken together,

these genetic studies have identified a large number of additional targets that

could be inhibited by adjuvants to improve the activity of various antibiotics.

In this section, I will focus on the work of Liu et al. who screened an E. coli

knockout collection of close to 4,000 strains, each lacking a different non-

essential gene (75). Their goal was to identify strains that were more

susceptible to a panel of 22 antimicrobials. In doing so, the authors

determined an antibiotic susceptibility profile for each mutant. Out of these

4,000 strains, they identified 283 that showed enhanced sensitivity to at least

one or more of the 22 tested antibiotics. These strains could be classified into

7 different categories based on the cellular function of the deleted gene

(Figure 8).

38

Figure 8. Functional categorisation of the E. coli K12 deletion mutants that are

hypersensitive to antibiotics. 283 E. coli strains showed increased sensitivity to at least

one or more of the 22 antibiotics. The strains were grouped into 7 different categories

based on the cellular function of the deleted gene. These include category 1: DNA

replication, recombination and repair, category 2: transport, efflux, cell wall and cell

membrane synthesis, category 3: protein synthesis, category 4: central metabolic

reactions, category 5: regulation, category 6: prophage-carried genes and cell adhesion,

and category 7: unassigned gene products. Data taken from (75). Reprinted with

permission.

Interestingly, 96 of the 283 strains (34 %) could be classified into the

category which is involved in transport, efflux, cell wall and cell membrane

synthesis (Figure 8), indicating that components of the cell envelope restrict

the action of many antibiotics. Table 1 shows the sensitivity profiles of each

of the 96 strains that belong to this category. Most of these mutants showed

enhanced sensitivity to several antibiotics. Strikingly, some of these mutants

such as the deletion strains acrA or tolC were more susceptible to nearly

all of the tested antibiotics (Table 1). The products of the two genes are

components of the multidrug efflux pump AcrAB-TolC. Namely, AcrA is

the periplasmic adapter protein of AcrAB-TolC whereas TolC represents the

OM efflux channel.

39

40

Table 1. Antibiotic sensitivity profiles of the 96 E. coli K12 knockout strains that are

involved in transport, efflux, cell wall and cell membrane synthesis. Sensitivity

profiles were categorized into three different groups with strong susceptibilities in dark

shades, medium susceptibilities in lighter shades and weak susceptibilities in lightest

shade. CIP, Ciprofloxacin; ENX, Enoxacin; NIT, Nitrofurantoin; MTR, Metronidazole;

SFX, Sulfamethoxazole; RIF, Rifampicin; GEN, Gentamicin; TOB, Tobramycin; NEO,

Neomycin; STR, Streptomycin; SPT, Spectinomycin; TET, Tetracycline; VAN,

Vancomycin; AMP, Ampicillin; RAD, Cephradine; FOX, Cefoxitin; ATM, Azetreonam,

CST; Colistin; CHL, Chloramphenicol; ERY, Erythromycin; FUS, Fusidic acid; TRI,

Triclosan. Large-scaffold antibiotics rifampicin, vancomycin and erythromycin are boxed

in red. Data taken from Liu et al (75). Reprinted with permission.

41

Since one of the goals of my doctoral thesis was to improve the activity of

those antibiotics which do not efficiently cross the OM, I was particularly

interested in the mutants that showed increased susceptibility to the large-

scaffold antibiotics rifampicin, vancomycin and erythromycin (Table 1).

Initially I focused on the glycopeptide antibiotic vancomycin. Vancomycin

is largely ineffective against Gram-negative bacteria because it does not

efficiently penetrate the OM. However, as shown in Table 2, there are 60

non-essential protein targets in E. coli whose inactivation improves the

activity of vancomycin. Notably, some of these mutants have previously

been shown to have increased sensitivity towards vancomycin. For example,

the hypersensitive mutant surA was 125-fold more sensitive to vancomycin

than the corresponding WT strain (Table 2). In addition, the surA mutant

became also sensitized to other large-scaffold antibiotics including

rifampicin and erythromycin (Table 1). SurA is a periplasmic chaperone that

is involved in the folding and transport of periplasmic proteins. Strains

lacking SurA are defective in the assembly of the OM, which results in an

increased sensitivity towards vancomycin and other antimicrobials (121,

122). Other mutants which have been previously described to be more

sensitive towards vancomycin include bamB and smpA ( bamE) (Table

2). They encode for the lipoproteins BamB and BamE which are part of the

BAM ( -barrel assembly machinery) complex. This complex catalyses the

folding and insertion of OMPs and is essential for the integrity of the OM

(123). Mutants lacking either BamB or BamE have a severe defect in the

OM which results in an increased sensitivity to various antibiotics including

vancomycin (124, 125).

42

Gene MIC

( g/ml)

Functional

category

Protein name

BW25113 wt 500 - -

surA 4 2 Peptidyl-prolyl cis-trans isomerase SurA

smpA 70 2 OM protein assembly factor BamE

bamB 100 2 OM protein assembly factor BamB

envC 100 2 Murein hydrolase activator EnvC

lpxL 100 2 Lipid A biosynthesis lauroyl acetyltransferase

tatC 100 2 Twin arginine protein translocation system

tolR 100 2 Colicin transport; Tol-Pal system component

ydcS 100 2 Putative ABC transporter periplasmic-binding protein YdcS

yciM 100 7 Lipopolysaccharide assembly protein B

recA 150 1 DNA strand exchange and recombination protein

envZ 150 2 Osmolarity sensor protein EnvZ

lpxM 150 2 Lipid A biosynthesis myristoyltransferase

nlpC 150 2 Probable endopeptidase NlpC

pal 150 2 Peptidoglycan-associated lipoprotein Pal

proW 150 2 Glycine betaine/L-proline transport system permease protein ProW

rfaC 150 2 Lipopolysaccharide heptosyltransferase I

ybgF 150 2 Periplasmic TolA-binding protein

yhdP 150 2 Uncharacterized protein YhdP

dnaK 150 2 Chaperone protein DnaK

hlpA 150 2 Periplasmic chaperone Skp

hscA 150 2 Iron-sulfur cluster biosynthesis chaperone HscA

hscB 150 2 Co-chaperone for iron-sulfur cluster biosynthesis

rimK 150 3 Ribosomal protein S6 modification protein

rlpA 150 3 Endolytic peptidoglycan transglycosylase RlpA

tufA 150 3 Translation elongation factor Tu 1

yfgC 150 3 -barrel assembly-enhancing protease

aceE 150 4 Pyruvate dehydrogenase E1 component

pgaC 150 4 Poly- -1,6-N-acetyl-D-glucosamine synthase

ycjU 150 4 -phosphoglucomutase

ygcO 150 4 Ferredoxin-like protein YgcO

dksA 150 5 RNA polymerase-binding transcription factor DksA

fur 150 5 Ferric uptake regulation protein

rsmF 150 5 Ribosomal RNA small subunit methyltransferase F

xapR 150 5 HTH-type transcriptional regulator XapR

yciT 150 5 Uncharacterized HTH-type transcriptional regulator YciT

ylcG 150 6 Uncharacterized protein YlcG

ydhT 150 7 Uncharacterized protein YdhT

recB > 150 1 RecBCD enzyme subunit RecB

ftsP > 150 1 Cell division protein FtsP

fepC > 150 2 Ferric enterobactin transport ATP-binding protein FepC

qmcA > 150 2 Protein QmcA

tatB > 150 2 Sec-independent protein translocase protein TatB

tonB > 150 2 Ton complex subunit TonB

yheL > 150 2 Sulfurtransferase complex subunit TusB

elaD > 150 3 Protease ElaD

rpmJ > 150 3 50S ribosomal protein L36

rpsO > 150 3 30S ribosomal protein S15

rrmJ > 150 3 Ribosomal RNA large subunit methyltransferase E

43

Table 2. List of E. coli K12 deletion strains which showed increased sensitivity to

vancomycin. Category 1: DNA replication, recombination and repair, category 2:

transport, efflux, cell wall and cell membrane synthesis, category 3: protein synthesis,

category 4: central metabolic reactions, category 5: regulation, category 6: prophage-

carried genes and cell adhesion, and category 7: unassigned gene products. Data taken

from (75). Reprinted with permission.

Interestingly, most of the remaining mutants in Table 2 had not been

previously described to show increased sensitivity towards vancomycin.

Notably, some of these mutants such as lpxL, lpxM, rfaC or yciM are

involved in LPS biosynthesis. Both LpxL and LpxM are acyltransferases

which transfer either a laurate or myristate chain onto the Kdo2-lipid A

precursor (126). RfaC (also known as WaaC) is a heptosyltransferase which

transfers the first heptose sugar onto the Kdo2 moiety of the LPS inner core

(127). YciM has been recently characterized as a modulator of LPS levels by

negatively regulating the biosynthesis of lipid A (128).

Based on the findings of this study, we reasoned that a small molecule which

inhibits any of the targets listed in Table 2 could be used as an antibiotic

adjuvant to enhance the activity of vancomycin. Since many vancomycin-

sensitive strains are also more susceptible to other antibiotics (Table 1), we

believe that such a small molecule inhibitor could potentially be used to

enhance the activity of several antibiotics. Hence, we carried out a high-

throughput screen in paper III to identify such small molecules.

yheM > 150 3 Protein TusC

yheN > 150 3 Sulfurtransferase TusD

rppH > 150 3 RNA pyrophosphohydrolase

cls > 150 4 Cardiolipin synthase A

cydB > 150 4 Cytochrome bd-I ubiquinol oxidase subunit 2

fdx > 150 4 2Fe-2S ferredoxin

ytjC > 150 4 Probable phosphoglycerate mutase GpmB

gpmM > 150 4 2,3-bisphosphoglycerate-independent phosphoglycerate mutase

iscS > 150 4 Cysteine desulfurase IscS

hns > 150 5 DNA-binding protein H-NS

ybgT > 150 7 Cytochrome bd-I ubiquinol oxidase subunit X

yjjY > 150 7 Uncharacterized protein YjjY

44

Summary of the papers

The overarching goal of my thesis was to understand how the OM barrier of

E. coli could be weakened. One approach that I took was to investigate the

feasibility of using small molecules to increase the permeability of the OM.

We reasoned that small molecules which permeabilize the OM could be used

as adjuvants or lead molecules for adjuvants to improve the activity of large-

scaffold antibiotics.

As a prelude to this, I initially investigated the periplasmic chaperone

network which is required for the biogenesis of OMPs. (Figure 9, left panel).

OMPs are synthesized on cytosolic ribosomes as precursors with an N-

terminal signal sequence and then transported across the IM by the SecYEG

translocase (129). After translocation and cleavage of the signal sequence,

newly exported OMPs are transported from the IM to the OM by the

periplasmic chaperone network SurA, Skp and DegP (130). The periplasmic

chaperone network, in particular SurA and Skp, is essential for the integrity

of the OM and strains lacking either of them are more sensitive to various

large-scaffold antibiotics including vancomycin (121, 122, 131).

45

Figure 9. Three major pathways required for the biogenesis of the OM in Gram-

negative bacteria. Left panel: OMPs are synthesized on cytosolic ribosomes and

subsequently translocated across the IM by the SecYEG translocase. Unfolded OMPs are

trafficked across the periplasmic space by the periplasmic chaperone network SurA, Skp

and DegP, and delivered to the -barrel assembly machinery (BAM) complex, which

folds and integrates OMPs into the OM. Middle panel: Lipoproteins are trafficked across

the IM via the SecYEG translocase and subsequently processed at the outer leaflet of the

IM. Processed lipoproteins destined to the OM are extracted from the IM by the ABC

transporter LolCDE, and then transferred to the periplasmic chaperone LolA. LolA

traffics lipoproteins across the periplasmic space to the OM lipoprotein LolB which

incorporates lipoproteins into the OM. Right panel: Lipopolysaccharide synthesis occurs

at the inner leaflet of the IM. Nascent LPS molecules are translocated across the IM by

the ABC transporter MsbA. Subsequently, LPS molecules are extracted from the IM by

the LptBCFG complex and then translocated across the periplasmic space via a

transenvelope bridge. Once arrived at the OM, LPS molecules are integrated into the OM

by the LptD-LptE complex. Adapted from (132). Reprinted with permission.

46

Before I joined the Daley lab, my former coworker Jörg Götzke identified a

protein with no annotated function, namely YfgM, which we thought might

be a novel member of this periplasmic chaperone network. He was able to

show that YfgM forms a complex with the periplasmic chaperone PpiD at

the SecYEG translocon (Figure 9, left panel). At this point, the function of

YfgM remained unknown. PpiD has been characterized as a member of the

periplasmic chaperone network, which assists in the release of newly

translocated OMPs proteins by preventing their premature aggregation (133,

134). Since YfgM interacts with PpiD at the SecYEG translocon, we

reasoned that YfgM might also be a part of this chaperone network.

In paper I, we had a closer look at the bacterial chromosome and found that

yfgM is located upstream of bamB. As previously mentioned, BamB is a

subunit of the BAM complex which is required for the integrity of the OM

(Figure 9, left panel). Strains lacking BamB have a defect in their OM and

thus are susceptible towards vancomycin. Hence, I was interested to

investigate if strains lacking YfgM also have a defect in their OM. To

address this question, we engineered strains that were lacking YfgM, PpiD

or other periplasmic chaperones such as SurA, Skp or DegP. We then

analyzed the integrity of the OM in these strains by performing an antibiotic

disc diffusion assay. In this assay, a filter disk containing a sub-inhibitory

concentration of vancomycin was spotted onto a lawn of bacteria (Figure

10A). Strains which have an intact OM, such as WT E. coli, are intrinsically

resistant towards vancomycin. Hence, no growth inhibition zone is observed

around the filter disk (Figure 10A, top panel). In contrast, strains with a

defect in their OM, such as surA or skp, have a compromised OM. In

these strains, vancomycin can access its target in the periplasmic space,

which results in growth inhibition around the filter disk (Figure 10A, lower

panel).

47

Figure 10. Disc diffusion assay to investigate the integrity of the OM. (A) WT E. coli

strains are intrinsically resistant towards vancomycin. Hence, no zone of growth inhibition is

observed around the filter disk (top panel). Strains with a defect in their OM, such as the

surA strain, became sensitized towards vancomycin which results in a growth inhibition

zone around the filter disk (bottom panel). (B) Disc diffusion assays were performed in WT E.

coli and various deletion strains to investigate the integrity of the OM. Figure 10B taken from

(135). Reprinted with permission.

Our data showed that deletion of the gene encoding for YfgM did not cause

any obvious defects in the OM (Figure 10B). In addition, we observed the

same phenotype for strains that were either lacking PpiD or DegP (Figure

10B). However, as previously reported, deletion of either SurA or Skp

destabilized the OM, which increased the sensitivity towards vancomycin

(Figure 10B).

Since periplasmic chaperones often have overlapping functions, their role in

the periplasmic chaperone network can only be elucidated by deleting them

in combination. For example, genetic deletion of ppiD and degP results in a

temperature-sensitive phenotype (134). Thus, we investigated if YfgM had

overlapping functions with other periplasmic chaperones. To address this

question, we engineered double knockout strains that were lacking YfgM in

48

combination with either PpiD, DegP, Skp or SurA. By using the vancomycin

susceptibility assay, we investigated the integrity of the OM in these strains

and found that deletion of YfgM in strains with either a ppiD or degP

background did not cause any obvious defects in the OM (Figure 10B).

However, deletion of YfgM in strains with either a skp or surA

background further compromised the integrity of the OM, as evidenced by

an increased growth inhibition zone around the filter disk containing

vancomycin (Figure 10B). Hence, these experiments suggest that YfgM is a

novel periplasmic chaperone which operates in the same network as Skp and

SurA.

In a follow-up study (paper II), we wanted to further understand the

function of YfgM by identifying its putative substrates. We hypothesized

that substrates of YfgM would be incorrectly folded or trafficked when

YfgM was absent from the cell, and therefore more prone to proteolytic

degradation. In this study, we used a comparative proteomic approach to

quantify the steady-state levels of proteins in strains lacking yfgM. Since our

previous study indicated that YfgM has overlapping functions with SurA and

Skp, we also included strains into the proteomic analysis that were lacking

yfgm together with either skp or surA, to exclude compensatory effects

caused by the major periplasmic chaperones.

Surprisingly, the proteomic analysis did not identify any changes in the

levels of OMPs or lipoproteins. However, we identified 9 IM proteins and 7

periplasmic proteins whose abundance was significantly changed. One key

finding was that a few proteins involved in the adaptation to gastrointestinal

stress (e.g. acid-resistance) were lower in abundance in strains lacking

YfgM. For example, the periplasmic chaperone HdeB which is involved in

the acid-stress response was lower in abundance in all three strains lacking

YfgM. To investigate if HdeB was misfolded / mistargeted in strains lacking

yfgM and thus turned over faster, we performed pulse-chase experiments. In

49

these pulse-chase experiments, we could show that HdeB was turned-over

faster in strains lacking yfgM. The identification of HdeB and other cell

envelope proteins as potential substrates of YfgM will be a valuable resource

for follow-up experiments that aim to decipher the function of YfgM.

Paper I + II provided novel insights into the periplasmic chaperone network

and how it contributes to the permeability of the OM. However, the findings

of these two studies do not provide any direct application that could be used

to improve the activity of antibiotics. In paper III our goal was to identify a

small molecule which causes the OM to be more permeable and thus might

be used as an antibiotic adjuvant. Initially, we wanted to identify an inhibitor

against the periplasmic chaperone SurA. Hence, we performed a high-

throughput screen in which we monitored growth of WT E. coli in the

presence of a sub-inhibitory concentration of vancomycin (150 μg ml-1) and

28,000 small molecules. We reasoned that a small molecule that inhibits

SurA would increase the activity of vancomycin, which would result in a

growth arrest. I established the assay conditions whereas the high-throughput

screen was performed by our collaboration partner from the Department of

Chemistry at Umeå University. The high-throughput screen identified one

promising molecule, namely MAC-13243. This small molecule has been

previously identified as an inhibitor of the periplasmic chaperone LolA

(136). LolA is an essential protein that functions as a periplasmic shuttle that

transports lipoproteins from the IM to the OM (Figure 9, middle panel).

All lipoproteins destined for the OM are synthesized in the cytoplasm as

precursors with an N-terminal sequence. After translocation through the

SecYEG translocon, lipoproteins are processed at the periplasmic side of the

IM, which involves sequential modification of a cysteine residue and

cleavage of the signal peptide by the lipoprotein-specific signal peptidase

LspA (63). Processed lipoproteins remain either attached to the IM or they

are extracted from the IM by the ABC transporter LolCDE (Figure 9, middle

50

panel). Subsequently, the periplasmic chaperone LolA captures lipoproteins

from the LolCDE complex and shuttles them across the periplasmic space to

the OM. At the OM, lipoproteins are transferred from LolA to LolB, which

localizes them to the OM.

Since inhibition of LolA by MAC-13243 leads to decreased levels of OM

lipoproteins (136), we hypothesized that this might affect the integrity of the

OM. To explore this possibility, I established an NPN (N-phenyl-1-

naphthylamine) uptake assay to investigate if the small molecule MAC-

13243 could be used to increase the permeability of the OM. The NPN

uptake assay is a commonly used method to analyze the integrity of the OM

(137). For example, WT E. coli strains have an intact OM and thus the

hydrophobic dye NPN cannot efficiently cross the OM. However, when the

OM is damaged, NPN can access phospholipids in the IM and the inner

leaflet of the OM, which results in prominent fluorescence. Using this assay,

I demonstrated that MAC-13243, when used at sub-inhibitory

concentrations, permeabilizes the OM of WT E. coli in a concentration-

dependent manner (Figure 11).

Figure 11. NPN uptake in WT E. coli cells. Left panel: WT E. coli cells were exposed to

different concentrations of MAC-13243 and NPN uptake was monitored. Right panel: The

increase in fluorescence was considered to be due to increased permeability of the OM

since the amount of MAC-13243 did not reduce cell viability. Abbreviations: MIC =

Minimal Inhibitory Concentration. Figure taken from paper III.

51

Since MAC-13243 permeabilized the OM of WT E. coli cells, I was curious

to investigate if MAC-13243 could also be used as an adjuvant for

antibiotics which do not efficiently cross the OM. To address this question, I

performed a series of checkerboard assays. The checkerboard assay is a

commonly used method to evaluate interactions between two drugs. In brief,

I found that MAC-13243 worked synergistically with either erythromycin or

novobiocin, meaning that there was added benefit when those two drugs

were used in combination (Figure 12).

Figure 12. MAC-13243 works synergistically with various large-scaffold antibiotics.

Heat plots illustrate inhibition of growth of WT E. coli in the presence of MAC-13243

and vancomycin, rifampicin, erythromycin or novobiocin. Growth percentage of E. coli is

shown with different colours where black represents 100% growth and red 0% growth.

Figure taken from paper III.

At this point, it remained unclear if the leaky phenotype, which we observed

after treating WT E. coli cells with MAC-13243, was caused by inhibition of

LolA. Since MAC-13243 is not stable in aqueous solution and a structural

analogue of its degradation product S-(4-chlorobenzyl)isothiourea, namely

A22, has been reported to inhibit the eukaryotic actin-homolog MreB

(Figure 17), it remained unclear what caused the cells to become more

permeable (138). To address this question, I used the CRISPRi system to

reduce the intracellular levels of LolA and showed that partial depletion of

52

LolA was sufficient to induce the permeable phenotype, as evidenced by an

increased permeability of the OM.

To summarize this study, we showed that inhibition of lipoprotein trafficking

could be an attractive target for small molecules. By performing a high-

throughput screen, we identified an inhibitor of the periplasmic chaperone

LolA, namely MAC-13243, and showed that this small molecule can be used

as an adjuvant to improve the activity of certain large-scaffold antibiotics.

In a parallel approach, we have collaborated with the Widmalm group from

the organic chemistry section at Stockholm University. Our common goal

was to break the OM permeability barrier by using small molecules which

target the synthesis of LPS (paper IV). Previous to this study, the Widmalm

group identified 3 small molecules in a fragment-based screen for inhibitors

against the glycosyltransferase WaaG (Figure 13A) (139). These molecular

scaffolds, namely L1-L3, have low affinity for WaaG and compete with its

natural substrate UDP-Glc for binding. This was of particular interest for us

since WaaG is a key enzyme in the synthesis of LPS. The peripherally

attached IM protein WaaG adds the first glucose residue to the outer core of

the growing LPS molecule (Figure 6).

Figure 13. Small molecular fragments L1-L3 and schematic representation of the

glucose transfer by WaaG. (A) L1, 4-(2-amino-1,3-thiazol-4-yl)phenol; L2, 4-(1H-

pyrrol-1-yl)benzoic acid; L3, 2-(1H-pyrrol-1-ylmethyl)pyridine; (B) The

glycosyltransferase WaaG transfers a 14C-glucose residue from UDP-Glc* to the waaG

LPS acceptor molecule. Figure 13A taken from (142). Reprinted with permission.

53

WaaG is essential for the stability of the OM and deletion of the gene

encoding for WaaG results in an inability to synthesize the outer core and the

O-antigen (Figure 14) (140). As a consequence, strains lacking WaaG

become more sensitive to several antibiotic classes (Table 1, formerly known

as rfaG) (75). We hypothesized that a small molecule that inhibits WaaG

could be used as an antibiotic adjuvant. Thus, in paper IV I explored if any

of these small molecular scaffolds could be used to inhibit WaaG in vitro. I

developed an activity assay for WaaG using 14C-labeled UDP-glucose and

LPS that had been purified from a waaG strain (Figure 13B).

Figure 14. Molecular dynamics simulations of OmpF trimer intercalated in OMs of E.

coli. OmpF trimer was intercalated in either E. coli rough LPS (left panel), E. coli K12 core

LPS (middle panel) or E. coli R1 core LPS with five repeating units of O6-antigen (right

panel). Lipid A is illustrated as pink spheres. Core sugars (gray) and O-antigen

polysaccharides (orange) are illustrated as stick models. The inner leaflet consists of PPPE

(blue spheres), PVPG (orange spheres), and PVCL2 (magenta spheres). Ca2+ ions are depicted

as cyan small spheres, K+ ions as green small spheres and Cl- ions as magenta small spheres.

Abbreviations: PPPE, 1-palmitoyl(16:0)-2-palmitoleoyl(16:1cis-9)-

phosphatidylethanolamine; PVPG, 1-palmitoyl(16:0)-2-vacenoyl(18:1 cis-11)

phosphatidylglycerol; PVCL2, 1,10-palmitoyl-2,20-vacenoyl cardiolipin. Figure taken from

(141). Reprinted with permission.

54

When I started to develop the assay for WaaG, I initially had problems with

its low activity. Since WaaG is a peripherally attached IM protein, I explored

if its activity could be improved by the addition of various lipids. After

several rounds of optimization, I discovered that I could improve the activity

of WaaG by including two E. coli membrane lipids, namely PG and CL, and

the non-ionic detergent CHAPS to the reaction mixture. Using these

optimized in vitro conditions, I showed that one of these small molecular

scaffolds, namely L1, is a weak inhibitor of WaaG with an IC50 of ~ 1 mM

(Figure 15).

Figure 15. L1 inhibits WaaG in vitro. Left panel: Mixed micelles consisting of 20 mM

CHAPS, 10 mM PG and 1 mM CL were added to LPS-TRUNC and UDP-Glc*. The reaction

was initiated by adding WaaG and incubated either with 2.5% DMSO or 25 mM of ligand

L1, L2 or L3. Samples were collected after various time points and inactivated by using

Laemmli buffer. Then, samples were separated by SDS-PAGE and detected by

autoradiography. Right panel: Relative activity of WaaG in the presence of either DMSO

or 25 mM L1, L2 or L3. Figure taken from (142). Reprinted with permission.

The observation that L1 could inhibit WaaG in vitro was an interesting

finding. Such small molecular fragments with low affinity for their target

(KD’s from high M to low mM) often represent a good starting point for the

55

design of a high-affinity inhibitor (143, 144). In this process, also known as

fragment-based drug design, small molecular fragments with low affinity for

their target are chemically elaborated or linked to produce a high affinity

inhibitor. Since L1 could be used to inhibit WaaG in vitro, we expanded

chemical space around L1 (and also L2 - L3), and created a fragment-based

library that included an additional 17 small molecular fragments (Figure 16,

unpublished data). At this point, the aim was to identify additional small

molecular fragments that either compete with the natural substrate UDP-Glc

for binding or inhibit WaaG in the in vitro activity assay.

Figure 16. Small molecular scaffold library L1-20.

56

Hence, I tested all small molecular fragments in the in vitro activity assay to

evaluate their inhibitory activity. Surprisingly, the results indicated that none

of the additional fragments had inhibitory activity (data not shown). These

findings were surprising since some of these fragments are structurally

closely related to L1 (Figure 16). The Widmalm group is currently

investigating by NMR spectroscopy if any of the additional small molecular

fragments compete with the natural substrate UDP-Glc for binding. Our

preliminary results indicate that one of these fragments, namely L8, also

binds to WaaG (data not shown). Despite the fact that the expanded library

did not contain a more potent inhibitor than L1, we could identify at least

one additional small molecular fragment that binds to WaaG. This provides

further insight for the design of a potent inhibitor against WaaG.

57

Conclusions and future perspectives

Gram-negative bacteria have developed sophisticated mechanisms to protect

themselves against noxious molecules such as antibiotics. Two of the major

mechanisms that limit the activity of many antibiotics include active efflux

by efflux pumps and reduced uptake across the OM barrier. The presented

doctoral thesis had the objective to investigate how the reduced uptake

across the OM barrier could be improved by destabilizing the OM. To

address this problem, I initially started investigating the periplasmic

chaperone network and how it contributes to the permeability of the OM

(paper I). In this paper, we identified a novel component of the SecYEG

translocon, namely YfgM, and showed that it operates in the same pathway

as the periplasmic chaperone network SurA/Skp. However, YfgM plays only

a minor role in this network since strains lacking YfgM did not have any

obvious defects in the OM. The molecular function of YfgM remains to be

determined but we speculate that it might act as a docking platform for the

periplasmic chaperones SurA/Skp. Interestingly, the periplasmic domain of

YfgM contains tetratricopeptide repeat (TPR) domains whose function has

not been determined yet. These domains are often involved in protein-

protein interactions and thus it might be interesting to further investigate the

function of the TPR domains in YfgM (145). There is also evidence that Skp

interacts with OMPs during their early translocation through the SecYEG

translocon (146). Hence, it is possible that Skp might dock to the SecYEG

translocon or a protein in close vicinity to it.

To better understand the function of YfgM, we performed a comparative

proteomic approach to identify potential substrates of YfgM (paper II). We

hypothesized that strains lacking YfgM might have a changed OMP profile

58

since YfgM operates in the same network as SurA/Skp. Although we did not

observe any significant changes in the levels of OMPs and lipoproteins in

strains lacking yfgM, the proteomic data revealed an unexpected insight into

the physiological role of YfgM. We identified a number of proteins that are

involved in acid-stress response, which were lower in abundance in strains

lacking YfgM. These findings were unexpected since YfgM had not been

shown to be induced during acid-stress previously (147–150). In an

additional experiment, we could confirm that strains devoid of YfgM had a

decreased survival rate at low pH. At this stage, it remains unclear if the

decreased survival rate at low pH in strains lacking YfgM is caused by a

defect in the cell envelope or by another secondary effect.

To address the reduced uptake of antibiotics across the OM, we performed a

high-throughput screen to look for small molecules that destabilize the OM

(paper III). In this study, we identified an inhibitor of the periplasmic

chaperone LolA, named MAC-13243. This small molecule had been

previously identified as a novel antimicrobial lead (136). However, I could

show that MAC-13243 can also be repurposed to an antibiotic adjuvant. I

observed that MAC-13243 synergized with the large-scaffold antibiotics

novobiocin and erythromycin, but not with vancomycin and rifampicin. At

this point, it remains unclear to us why MAC-13243 works synergistically

with some large-scaffold antibiotics but not with others. It is worth noting

that Krishnamoorthy et al. reported that the activity of these 4 antibiotics is

significantly limited by the OM and/or by active efflux (72). Surprisingly,

they found that the OM presented no major obstacle for novobiocin whereas

active efflux drastically reduced its activity. In the case of rifampicin and

vancomycin, they found that the OM barrier significantly limited their

activity whereas inactivation of active efflux only minor improved their

activity. In the case of erythromycin, a combination of both reduced uptake

and active efflux limited its activity.

59

Despite the preliminary findings, I believe that MAC-13243 has certain

limitations in its current form that prevent it from being used as an antibiotic

adjuvant. First of all, the synergistic interactions between MAC-13243 and

either novobiocin or erythromycin were moderate and only observed under

specific conditions. One of the major limitations of MAC-13243 might be its

limited stability in aqueous solution (151). MAC-13243 degrades in aqueous

solution into one molecule of 3,4-dimethoxyphenethylamine, two molecules

of formaldehyde and one molecule of S-(4-chlorobenzyl)isothiourea (Figure

17A). Thus, an attempt to improve the chemical stability of MAC-13243

could improve its activity as an antibiotic adjuvant. This could be achieved

by stabilizing the central triazine ring of MAC-13243, which is prone to

hydrolysis (151).

Figure 17. Degradation of MAC-13243 in aqueous solution. (A) MAC-13243 is

degraded into one molecule 3,4-dimethoxyphenethylamine, two molecules of

formaldehyde and one molecule S-(4-chlorobenzyl)isothiourea (151). Both MAC-13243

and the degradation product S-(4-chlorobenzyl)isothiourea bind to LolA (151) (B) An

analogue of the degradation product, named A22 or S-(4-dichlorobenzyl)isothiourea, has

been shown to bind to LolA. Interestingly this compound is an inhibitor of the

cytoskeletal protein MreB (138). Figure adjusted from (151). Reprinted with permission.

60

Interestingly, one of the degradation products of MAC-13243, namely S-(4-

chlorobenzyl)isothiourea, is a structural analogue of compound A22, which

has been identified as an inhibitor of the cytoskeletal protein MreB (Figure

17B) (138). Taylor et al. showed that A22 works synergistically with both

novobiocin and rifampicin in E. coli (115). Hence, the design of a molecule

which inhibits both LolA and MreB could be an interesting concept to

further enhance the synergistic effects of MAC-13243.

I personally think that it would be most interesting to expand on the findings

of paper IV. In this paper, I showed that the small molecular fragment,

namely compound L1, can be used to inhibit the glycosyltransferase WaaG

in vitro. However, due to its weak inhibitory activity, L1 is not a molecule

that can be used in its current form as an antibiotic adjuvant. However, we

believe that L1 represents a good lead fragment for the design of a more

potent inhibitor against WaaG. By expanding chemical space around L1, we

could identify an additional molecule, namely L8 (Figure 16), that binds to

WaaG in vitro. In a next optimization round, we plan to systematically

evaluate the chemical space around these two molecules to gain further

insight into the properties that are required for binding to and inhibiting

WaaG.

61

Populärvetenskaplig sammanfattning

Antibiotika anses vara en av de viktigaste upptäckterna inom

humanmedicinen. Sedan de infördes in i klinikerna på 1940-talet har de

framgångsrikt använts för att bota bakteriella infektioner. På grund av sin

omfattande användning och missbruk under de senaste decennierna har dock

många bakteriearter som inte längre är känsliga mot de flesta antibiotika

uppstått. Dessa så kallade multiresistenta bakterier har utvecklat olika

tillvägagångssätt för att inaktivera antibiotika. Följaktligen har vi ont om

behandlingsalternativ och är ofta tvungna att använda äldre antibiotika som

är mindre säkra.

Resistens mot antibiotika är ett allmänt problem. Gramnegativa bakterier är

dock arter som är särskilt bekymmersamma. Dessa bakterier dödar tusentals

människor årligen och anses därför vara ett stort hot mot folkhälsan. Ett av

de största problemen är att många antibiotika är ineffektiva mot

gramnegativa bakterier. Detta beror dels på en fysisk barriär, nämligen det

yttre membranet, som hämmar effektivt upptag av många antibiotika.

I den presenterade doktorsavhandlingen undersökte jag hur dessa fysiska

barriärer kunde försvagas. Det ultimata målet med min avhandling var att

hitta små molekyler som skulle kunna användas för att bryta denna fysiska

barriär. Vi resonerade att dessa små molekyler skulle kunna användas för att

förbättra upptagningen av de antibiotika som normalt inte effektivt passerar

yttermembranet.

62

Acknowledgements

I would like to take the opportunity to thank my supervisor Daniel Daley for his excellent supervision during the past 5 years. I really enjoyed it to be part of your group. Thanks mate! I also would like to thank our current group members Kiavash, Rageia, Zoe and Patrick, and the former group members Jörg, Stephen, Bill and Isolde. It was very pleasant to work with all of you and I think we have a really good ambience in the group. Special thanks to my co-supervisor Göran Widmalm and his current group members Jonas and Alessandro. It was fun to work together with you guys! Also a special thanks to our former collaborators Klaas, Spyridon, Amin, Olof and Åke. I am also grateful to my former students Lars Nilsson, Johanna Hultgren, Muna Amanuel, Andreas Dunge and Anja Blümler. Thanks to our neighbors from the Jan-Willem de Gier group: Thomas, Zhe, Henry and Alex. Special thanks to my table soccer friend Thomas for never giving up and Alex the Greek for all the insightful philosophical discussions. Thanks to the Rob Daniels group: Henrik, Hao, Dan, Rebecca and the former group members Johan and Diogo. It was always nice to see other people in the lab on the weekends. A big thanks to the Gunnar von Heijne and Ingmarie Nilsson group: Renuka, Grant, Felix, Ioanna, Luigi, Nir, José, Nina and Åsa. Special thanks to my bongo friend Mr. Felix – it was a pleasure to explore Africa with you! Thanks to the Tara Hessa group: Tara, Theresa, Genia and Anastasia. Cool group, good people!

63

Thanks to the Elzbieta Glaser group: Pedro and Beata for always being in a good mood and Elzbieta for never-ending but interesting discussions at the lunch table. Many thanks to Brzezinski and Ädelroth group members Jacob, Johan, Tobias, Camilla, Ingrid, Max, Nathalie and Pia. Great thanks to my climbing and sailing friend Max (aka sailor man) – I knew it was a good decision to have a friend with a sailing boat! We truly had some good laughs and I will never forget the large number of fish we caught - zero! Thanks to the David Drew group members Emmanuel, Mathieu, Aziz, Povilas, Pascal and Magnus. Bsunderä Dank äm bärtigä Päsci und sim Schätzi dr Regula - heb sorg zunnärä! Du bisch wahrhaftig en Glückspilz! Sogar dr Andi us Rümlang wär nidisch! Thanks to the Martin Ott group members Hannah, Mama, Magdalena, Tamara and Braulio for the discussions about the book exam and other stuff. Special thanks to Alex Tuuling, Maria Sallander, Anita Hjelm, Lotta Sturell, Karin Häggbom Sandberg, Malin Kamb, Håkan Thorén, Matthew Bennett, Ann-Britt Rönnell and Peter Nyberg for running DBB in the background. Last but not least, a very special thanks to my family and friends back home for their great support and visits to Stockholm. Looking forward to see you again!

64

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