scientific meeting about clinical proteomics. 2nd...

SCIENTIFIC MEETING ABOUT CLINICAL PROTEOMICS. 2nd EDITION. H it l U i it i L P 18 19 N b

Proteomics applied to microbiology and infectious diseases

Hospital Universitario La Paz, 18-19 November

Proteomics applied to microbiology and infectious diseases.Advances in Proteomics.

"Candida albicans host interaction: insights from proteomics"

Dpto. de Microbiología IIUnidad de ProteómicaUnidad de Proteómica Facultad de Farmacia, UCM. Madrid

OUTLINE

Why proteomics to study host-pathogen interaction ?

• To discover proteins of clinical interest: diagnostic and prognostic markers and vaccine candidates

Immunoproteomics with sera of patients with invasive candidiasis (IC)

• To discover new virulence factors and to know which aspects of thei ibl f th d t ti f th thimmune response are responsible for the destruction of the pathogen

Candida albicans –macrophagesp g

COMENSALISM

CANDIDA HOST INTERACTIONAN OVERVIEW OF THE CURRENT SETTING FOR HUMAN CANDIDIASIS C. albicans, can cause host damage

Time

Dam

age

COMENSALISM

BIOLOGICAL INTERESTAlteration of

by mechanisms mediatedboth by host (predisposing factors) and by fungus (virulence factors)

INTERACTIONDimorphism

Phenotypic switching

Extracellular hydrolases

AdhesionVirulence

factors FUNGUS Predisposingfactors

Immune response impairment

microfloracomposition

HOST Natural barrier disruption

PROTEOMICS?RO

TEO

MIC

S?

Depending on the

SuperficialChronic mucocutaneous m

age

CANDIDIASIS

Others?impairmentPROTEOMICS?

PR

Depending on theunderlying host defect, C.albicans can cause different types ofinfections

SystemicTime

Dam

No signs andDiagnosis

No rapid and accurate tests

Innate

Acquired

Host immune response

PMN leukocytes

Cellular

Macrophages

PRO

TEOM

IC

Due to the host’sDifficulties

No signs and symptoms

TreatmentSide-effects

Resistance

AcquiredHumoral

Reduced efficacy

CS?

New

Due to the host sdebilitated immune response, together with difficultiesin the diagnosis and treatment the outcome

PROTEOMICS?Time

Dam

age

ERADICATIONPROTEOMICS? diagnostic and therapeuticstrategies

Time

Dam

age

DEATHtreatment, the outcome of these patients is often fatal

CLINICAL INTERESTPitarch, A., Nombela C. and Gil C. “Microbial proteomics: Functional Biology of whole organisms”. Wiley CH, 2006.

To discover proteins of clinical interest:

diagnostic and prognostic markers and vaccine

didcandidates

IMMUNOPROTEOMICS

Pitarch et al., Molecular &Cellular Proteomics. 2006

DIFFERENTIAL IMMNUNOPROTEOMICS IS A SUCCESFUL APPROACH FOR STUDYING THE SEROLOGICAL RESPONSE

THE Candida IMMUNOME

APPROACH FOR STUDYING THE SEROLOGICAL RESPONSE TO THE CANDIDA IMMUNOME (2D , WB AND MS)

SAMPLE PREPARATION (C. albicans proteins)

2-D imaging film

Sera from patients

Group 1(e.g., Healthy

individuals’ sera)

Group 2(e.g., Systemic

candidiasis patients’ sera)IEF SDS

Mr84

+-

2-D gel 2-D blot

g g

Murine(pre)immune sera

or

with/without systemic candidiasis

4 5 6 7 pI

60483530

15

SEPARATION of C. albicans proteins

by 2-DE

DETECTION of C. albicans immunogenicproteins by WESTERN-BLOT and

DEFINITION of“IMMUNE PROTEOMES”

4 5 6 7 p

ELECTROBLOTTINGof 2-D gels onto nitrocellulosemembranes

·

DIFFERENTIAL DISPLAY and QUANTIFICATION by bioinformatic analysis

EXCISION and “IN GEL” DIGESTION of immunogenic

protein spots of interest Discovery Phase

nsity

100

p p

P < 0.0001

vel


m/z1000 3500

Rel

ativ

e In

ten

0 Group 1 Group 2

Ant

ibod

y le

v

STATISTICAL ANALYSIS using clinical data

IDENTIFICATION of immunogenicproteins of interest by

MS and database searching

m/z1000 3500

Proteins of clinical interest

Group 1 Group 2

Th diff t tiDiagnostic markers?

Prognostic markers?

Vaccine candidates?

The different antigenrecognition patterns

are compared by bioinformatic analysis using clinical data

88 kDa

0h 24h 48h 72h 96h

analysis using clinical data

Proteins can be expressed as

recombinant proteinsHETEROLOGOUS EXPRESSION

of proteins of clinical interest

recombinant proteins

Validation or prototype

VALIDATION in routine assays(e g ELISA)

prototype development phase

(e.g., ELISA)

DETECTION OF ANTIGENS IN PATIENTS WITH INVASIVE CANDIDIASISTHE Candida IMMUNOME


WESTERN BLOTS WITH SERA FROM PATIENTS WITH INVASIVE CANDIDIASIS (45) AND SERA FROM CONTROL INDIVIDUALS (118)CANDIDIASIS (45) AND SERA FROM CONTROL INDIVIDUALS (118)

IMMUNOSUPRESSED PATIENTS

NON-IMMUNOSUPRESSED PATIENTS

Solid Tumor* Bronchopulmonary neoplasm

* Pancreas/colon adenocarcinoma

Nonmalignant diseases

* Respiratory dysfunction

* Gastrointestinal pathology* Bladder neoplasm

Hematological malignancy

* Leukaemia

Gastrointestinal pathology

* Others

* Lymphoma

* Myolodysplasia

* Multiple myeloma

DETECTION OF ANTIGENS IN PATIENTS WITH SYSTEMIC CANDIDIASISTHE Candida IMMUNOME

WESTERN BLOTS WITH SERA FROM PATIENTS WITH INVASIVE CANDIDIASIS (45) AND SERA FROM CONTROL INDIVIDUALS (118)

IMMUNOSUPRESSED PATIENTS NON-IMMUNOSUPRESSED PATIENTS

84

1

IEFSDS 84

IEF

SDS

1

60

1

60 Expression profiles were very heterogeneous

48

35 12

48

3512

4 4 5 5 5 5 pI

303-18 protein spots

6-50 protein spots4 4.5 5 5.5 p

pI4 4.5 5 5.5 6 7 7.5

Control sera were negative or recognized from 1 to 10 spots

BIOMARKER DISCOVERY IN THE WHOLE SOLUBLE Candida PROTEOME


SOLUBLE Candida PROTEOME

42 different C. albicansproteins becomeproteins become targets of the human immune responseimmune response to systemic candidiasis35 f th35 of them arenew antigens

Pitarch et al., 2004. Proteomics

Cytoplasmic extracts

BIOMARKER DISCOVERY IN THE Candida CELL WALL IMMUNOME (proteins secreted by protoplast in active wall regeneration)


(proteins secreted by protoplast in active wall regeneration)


Y t t l tY t t l tYeast protoplastsYeast protoplasts


Yeast protoplasts after 2 hours of regenerating the cell wall


WALL ASSOCIATED PROTEINS (PROTEINS SECRETED

IEF

WALL ASSOCIATED PROTEINS (PROTEINS SECRETED BY PROTOPLASTS IN ACTIVE WALL REGENERATION)

11684

Mr

(kDa)

IEFSDSMet6p

84

60

48Gap1p

Eno1p Pgk1p

Metabolic proteins35

28Bgl2p

Fba1pBgl2, glucan 1,3-β-

glucosidase

1528

4 4 5 5 5 5 6 6 5 7 7 5 8 pITpi1p

g

4 4.5 5 5.5 6 6.5 7 7.5 8 p

Pitarch et al., 2006. Molecular & Cellular Proteomics

DISTRIBUTION OF SERUM LEVELS OF IgG ANTIBODIES


LEVELS OF IgG ANTIBODIES AGAINST Candida WALL ASSOCIATED PROTEINS

Their distributiondiffered significantlybetween IC patientsand controls

Pitarch et al., 2006. Molecular & Cellular Proteomics


IT WAS POSSIBLE TO DISCRIMINATE IC PATIENTS FROM CONTROLSIT WAS POSSIBLE TO DISCRIMINATE IC PATIENTS FROM CONTROLS. THIS STUDY HIGHLIGHTED THE HETEROGENICITY OF THEIR EXPRESSION PROFILES Pitarch et al., 2006. Molecular & Cellular Proteomics

WHICH ANTIBODIES ARE PREDICTORSTHE Candida IMMUNOME

OF INVASIVE CANDIDIASIS?

Multivariate logistic regression models demonstrated that high levels of antibodies against glucan 1,3-β-glucosidase (Bgl2p) and the anti-wall phophoglycerate kinase antibody seropositivity were the only predictors of IC.

Receiver operating characteristic curve analysis revealed no differences between their combined evaluation and measurement of anti-Bgl2p antibodies alone.

Anti Bgl2 IgG antibodies are novel independent biomarkers for early IC diagnosis (78 % sensitivity and 93 % specificity)

ALTERNATIVE STRATEGIES FOR THE IDENTIFICATION


ALTERNATIVE STRATEGIES FOR THE IDENTIFICATION OF CELL SURFACE PROTEINS

CW

PM

SURFOMEProteins located on the outermost layer of the cell wall.


Cell integrity assay

Cell surface digestion

Peptide separation

Cell surface digestion

Peptide separation

y

Pellet

Cell integrity assayProtein identification Protein identification

containing cells and debris

SupernatantTryptic digestion

in 5 mM DTT Centrifugation109 cells p

containing tryptic peptides

in 5 mM DTT

37 ºC5 -30 min

3000xg5 min

Peptide separation by Nano-HPLC

and identification in a

Trypsin

TrypControl Trypsinl

DTTl MALDI-TOF-TOF

Surface exposed proteins

+ DTT(no Tryp, no DTT) onlyonly

Raquel Raquel MartínezMartínez--LópezLópez et al., et al., 2008. Proteomics. 2008. Proteomics. HernáezHernáez et al., et al., 2010, Journal of Proteomics2010, Journal of Proteomics

THE Candida IMMUNOMESURFOMEBIOGENESIS OF CELLULAR COMPONENTS

Cell wall organization and biogenesis9 6 10 8 |CA0156| Scw1p glucanase (by homology)5 6 5 4 |CA0822| Exg1p glucan 1,3-beta-glucosidase

5 10 15 30 min

5 6 5 4 |CA0822| Exg1p glucan 1,3 beta glucosidase4 3 3 4 |CA0883| Sun41p Putative cell wall beta-glucosidase (by homology)3 3 3 3 |CA1541| Bgl21p endo-beta-1,3-glucanase (by homology)2 2 2 4 |CA0375| Crh11p Probable membrane protein (by homology)0 1 1 1 |CA5213| Ssr1p Secretory Stress Response protein 1 (by homology)0 1 1 1 |CA0605| Utr2p 1,3-1,4-beta-glucanase (by homology)| | p , , g ( y gy)0 0 1 0 |CA3867| Phr2p pH-regulated protein 25 4 5 4 |CA2303| Tos1p putative Anchor subunit of a-agglutinin (by homology)1 1 1 1 |CA1678| IPF5185 putative cell wall protein (by homology)2 0 0 1 |CA4800| IPF8796 putative GPI-anchhored protein related to Phr1, Phr2 and Phr34 4 6 6 |CA5091| Acf3p endo-1,3-beta-glucanase| | p , g4 2 2 3 |CA5987| Cht3p chitinase 3 precursor1 1 2 1 |CA1053| Scw11p glucanase gene family member, 3-prime end (by homology)3 3 4 4 |CA5344| Pry2p putative pathogen related proteins (by homology)1 1 1 1 |CA1051| Cht2p chitinase 2 precursor

Citoskeleton organization and biogenesis0 0 2 0 |CA5255| Act1p actin (by homology)

CELL RESCUE, DEFENSE AND VIRULENCE6 6 5 5 |CA5232| Sun42p Putative cell wall beta-glucosidase (by homology)0 1 3 0 |CA4336| Ddr48p stress protein (by homology)3 1 2 2 |CA1576| Hyr1p hyphally regulated protein0 3 4 2 |CA2857| Ssa1p Heat shock protein of HSP70 family1 1 1 1 |CA3534| Ssb1p heat shock protein 700 0 1 0 |CA2830| Rbt1p repressed by TUP1 protein 12 2 3 3 |CA0104| Rbt4p repressed by TUP1 protein1 1 1 1 |CA0909| Als1.5p agglutinin-like protein, 5-prime end1 0 1 0 |CA1527| Als4.5p agglutinin-like protein, 5-prime end

THE Candida IMMUNOMESURFOMEMETABOLISM

5 10 15 30 minMETABOLISMMETABOLISM

3 3 3 3 |CA1691| Pgk1p Phosphoglycerate kinase0 1 0 1 |CA3874| Eno1p Enolase I (2-phosphoglycerate dehydratase)0 0 0 1 |CA4765| Adh1p alcohol dehydrogenase (by homology)2 1 2 2 |CA2474| Pdc11p Pyruvate decarboxylase (by homology)0 0 1 1 |CA2162| Pdb1p pyruvate dehydrogenase (by homology)0 0 1 0 |CA5239| Gnd1p 6-phosphogluconate dehydrogenase

METABOLISM3 3 3 3 |CA1691| Pgk1p Phosphoglycerate kinase0 1 0 1 |CA3874| Eno1p Enolase I (2-phosphoglycerate dehydratase)0 0 0 1 |CA4765| Adh1p alcohol dehydrogenase (by homology)2 1 2 2 |CA2474| Pdc11p Pyruvate decarboxylase (by homology)0 0 1 1 |CA2162| Pdb1p pyruvate dehydrogenase (by homology)0 0 1 0 |CA5239| Gnd1p 6-phosphogluconate dehydrogenase0 1 1 0 |CA2858| Acs2p acetyl-coenzyme-A synthetase (by homology)0 2 0 1 |CA1983| Ilv5p ketol-acid reducto-isomerase (by homology)1 1 0 0 |CA0959| Sam2p S-adenosylmethionine synthetase 20 0 1 1 |CA0653| Met6p 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase3 0 5 2 |CA5892| Gap1p Glyceraldehyde-3-phosphate dehydrogenase

PROTEIN FATE

0 1 1 0 |CA2858| Acs2p acetyl-coenzyme-A synthetase (by homology)0 2 0 1 |CA1983| Ilv5p ketol-acid reducto-isomerase (by homology)1 1 0 0 |CA0959| Sam2p S-adenosylmethionine synthetase 20 0 1 1 |CA0653| Met6p 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase3 0 5 2 |CA5892| Gap1p Glyceraldehyde-3-phosphate dehydrogenase

PROTEIN FATEPROTEIN FATE0 0 0 1 |CA0915| Kar2p dnaK-type molecular chaperone (by homology)

Protein Synthesis1 1 1 1 |CA2818| Rpl13p Ribosomal protein1 1 2 1 |CA0615| Rpl3p 60S large subunit ribosomal protein L3.e (by homology)0 1 1 1 |CA0736| Rpl20Bp ribosomal protein (by homology)1 1 1 1 |CA1440| Rpl17Bp RPL17B ribosomal protein L17.e1 0 1 1 |CA4225| Rpl39 3p ribosomal protein L39 3 prime end

PROTEIN FATE0 0 0 1 |CA0915| Kar2p dnaK-type molecular chaperone (by homology)

Protein Synthesis1 1 1 1 |CA2818| Rpl13p Ribosomal protein1 1 2 1 |CA0615| Rpl3p 60S large subunit ribosomal protein L3.e (by homology)0 1 1 1 |CA0736| Rpl20Bp ribosomal protein (by homology)1 1 1 1 |CA1440| Rpl17Bp RPL17B ribosomal protein L17.e1 0 1 1 |CA4225| Rpl39 3p ribosomal protein L39 3 prime end1 0 1 1 |CA4225| Rpl39.3p ribosomal protein L39, 3-prime end0 1 1 1 |CA3539| Rps22p ribosomal protein by homology0 1 1 0 |CA4562| Rps8Ap ribosomal protein (by homology)0 0 1 1 |CA4862| Efb1p translation elongation factor eEF1beta1 2 2 3 |CA2810| Eft2p translation elongation factor 22 3 3 2 |CA0362| Tef1p translation elongation factor eEF1 alpha-A chain

1 0 1 1 |CA4225| Rpl39.3p ribosomal protein L39, 3-prime end0 1 1 1 |CA3539| Rps22p ribosomal protein by homology0 1 1 0 |CA4562| Rps8Ap ribosomal protein (by homology)0 0 1 1 |CA4862| Efb1p translation elongation factor eEF1beta1 2 2 3 |CA2810| Eft2p translation elongation factor 22 3 3 2 |CA0362| Tef1p translation elongation factor eEF1 alpha-A chain

TRANSPORT0 0 0 1 |CA2300| Pma1p plasma membrane H+-transporting ATPase 10 0 0 1 |CA3415| Fre30.53p Strong similarity to ferric reductase, internal fragment (by homology)0 0 0 1 |CA2923| Fet32p2 cell surface ferroxidase (by homology)1 1 0 1 |CA5388| Pet9p ADP/ATP carrier protein (by homology)

UNKNOWN

TRANSPORT0 0 0 1 |CA2300| Pma1p plasma membrane H+-transporting ATPase 10 0 0 1 |CA3415| Fre30.53p Strong similarity to ferric reductase, internal fragment (by homology)0 0 0 1 |CA2923| Fet32p2 cell surface ferroxidase (by homology)1 1 0 1 |CA5388| Pet9p ADP/ATP carrier protein (by homology)

UNKNOWN2 1 1 2 |CA4381| IPF20169 unknown function0 2 1 2 |CA5714| IPF2431 similar to Saccharomyces cerevisiae Tsa1p thiol-specific antioxidant-like pro1 1 2 2 |CA0586| IPF13493 Unknown function2 2 2 1 |CA1345| IPF6003 similar to Saccharomyces cerevisiae Msb2p multicopy suppressor of a CDC1 1 0 0 |CA4642| IPF1500 unknown function

2 1 1 2 |CA4381| IPF20169 unknown function0 2 1 2 |CA5714| IPF2431 similar to Saccharomyces cerevisiae Tsa1p thiol-specific antioxidant-like pro1 1 2 2 |CA0586| IPF13493 Unknown function2 2 2 1 |CA1345| IPF6003 similar to Saccharomyces cerevisiae Msb2p multicopy suppressor of a CDC1 1 0 0 |CA4642| IPF1500 unknown function

Some of these proteins can be immunogenic and useful for diagnosis

RELIABILITY OF ANTIBODIES TO CANDIDA METHIONINESYNTHASE AND ENOLASE FOR DIAGNOSIS OF SYSTEMIC


SYNTHASE AND ENOLASE FOR DIAGNOSIS OF SYSTEMIC CANDIDIASIS

Expression vector used for TAP-rMet6p production in P. pastoris

Structure of the TAP-rMet6p

Pitarch et al. 2007.Proteomics Clin. ApplPitarch et al., 2008.Proteomics Clin Appl.

Diagnostic validation of the ELISA for measurement of serum anti-


gCandida Met6p and Eno1p IgG antibody concentrations

Distribution of serum anti-Eno1p Distribution of serum anti- pIgG antibody concentrations in SC and non-SC patients according to

their immune system status.

Eno1p IgG antibody concentrations among the

study groups

ENOLASE Specificity 91 % 96 % ; Sensitivity 74% -67 % (immunosupressed and immunocompetent, respectively)MET6 Specificity: 95-100 % , Sensitivity: 62-52 % (with and without iatrogenic immunosupression respectively)

PREDICTION OF THE CLINICAL OUTCOME IN INVASIVE CANDIDIASIS


PATIENTS BASED ON SERUM PROFILING OF ANTIBODY RESPONSE

Candida albicans total extracts

Poor PrognosisGood Prognosis oo og os sGood og os s

These profiles are too heterogeneous (31 Antigens)

TWO-WAY HIERARCHICAL CLUSTER ANALYSESOF GLOBAL ANTI-CANDIDA ANTIBODY-REACTIVITYPATTERNS IN IC PATIENTS

Serum samples

PATTERNS IN IC PATIENTS

IgG Poor Prognosis Good Prognosisgantibodies

Poor Prognosis Good Prognosis

Serum samples from IC survivors and IC non-survivorssegregated into two distinct groups

Pitarch et al., Molecular & Cellular Proteomics. 2010 (In Press)

segregated into two distinct groups

DEVELOPMENT AND VALIDATION OF IC PROGNOSTIC PREDICTOR

Met6p Ssb1p Gap1p Hsp90p Pgk1p

Cross-validation Independent validation

1

3

edic

tor p = 0.001 p = 0.001

-1

1

ostic

pre

-5

-3

C p

rogn

o

PoorPrognosis

GoodPrognosis

PoorPrognosis

GoodPrognosis

-7IC

Prediction strengths of IC prognostic predictorin cross-validation and in independent validation

A general scheme of clinical biomarker development for ICA general scheme of clinical biomarker development for IC.

We validated the IC prognostic predictor

VALIDATION OF IC PROGNOSTIC PREDICTOR BY AN IMMUNOASSAY BASED ON THE 5 ANTI- CANDIDA ANTIBODIES

TwoTwo--way hierarchical cluster analyses of the IC prognostic way hierarchical cluster analyses of the IC prognostic y y p gy y p gpredictor in testing IC patients. Serum samples from IC predictor in testing IC patients. Serum samples from IC survivors and IC nonsurvivors and IC non--survivors survivors segregated into two distinct segregated into two distinct groups validating thegroups validating the immunoproteomicsimmunoproteomics resultsresultsgroups validating the groups validating the immunoproteomicsimmunoproteomics resultsresults

CLINICAL BIOMARKERS FOR INVASIVE CANDIDIASISTHE Candida IMMUNOME

CLINICAL BIOMARKERS FOR INVASIVE CANDIDIASIS

• More than 40 C.albicans antigens were identified using immunoproteomics.

• A systematic proteomic approach coupled with bioinformatic analysis has shown that high levels of antibodies against glucan 1,3-beta-glucosidase (Bgl2p) were predictors of IC.

• We have expressed in Pichia pastoris and Escherichia coli some proteins of clinical interest: Bgl2p, Met6p, Eno1p, Hsp90p, Pgk1p, Ssb1p and Gap1p.

• Analytical and clinical validations of the levels of antibodies against some C. albicans proteins have been carried out.

• We have defined a 5-IgG antibody-reactivity signature for IC prognosis.Antibodies against Met6p, Hsp90p and Pgk1p (putative Candida virulence factors and anti-apoptotic mediators) are associated with good prognosis and p p ) g p gprotective patterns. Antibodies against Ssb1p and Gap1p (potential pro-apoptotic mediators) are associated with poor prognosis and risk patterns.

• The full potential of these antigens for diagnosis and/or follow up of IC is• The full potential of these antigens for diagnosis and/or follow-up of IC is currently being addressed by a large scale screening of patients’ and controls’ serum specimens.

T t d th h t thTo study the host-pathogen interaction:

C albicans macrophagesC.albicans-macrophages

EXPRESSION PROTEOMICS

Fernandez-Arenas et al., Molecular and Cellular Proteomics. 2007

EXPERIMENTAL DESIGNCANDIDA ALBICANS MACROPHAGE INTERACTION

CANDIDA HOST INTERACTION

CANDIDA ALBICANS – MACROPHAGE INTERACTION

GENOMICSPROTEOMICS RATIO 1:1PROTEOMICS RATIO 1:1

DNA MICROARRAYS1,5 and 3h. of interaction

DIFFERENTIAL PROTEIN EXPRESSION

3h. of interaction

PROTEIN IDENTIFICATIONCANDIDA HOST INTERACTION

78 PROTEIN SPOTS REPRESENTING 67 DIFFERENT67 DIFFERENT PROTEINS WERE IDENTIFIED BY MALDI TOF/TOF

SMS

(40 over-expressed and 27 under-and 27 underexpressed)


FUNCTIONAL CLASSIFICATION OF DIFERENTIALLY EXPRESSED PROTEINS (Data bases employed: PROTEOME, CANDIDA DB Y CGD)

UNCHARACTERIZED; 6,0%BIOGENESIS OF CELLULAR C compound and carbohydrate

METABOLISM53.7%

CELLULAR TRANSPORT; 6,0%

Assembley of protein Amino acid metabolism; 7 5%

BIOGENESIS OF CELLULARCOMPONENTS; 6,0%

C compound and carbohydratemetabolism; 17,9%

complexes; 3,0%Amino acid metabolism; 7,5%

Nucleotide metabolism; 9,0%

; Lipid and fatty acid β oxidation

Protein folding, modif ication anddegradation ; 16,4%

; Lipid and fatty acid β oxidation7,5%

Metabolism of vitamins; 3,0%

& Oxidative responseDetoxif ication; 10 4% ; Glyoxylate and TCA cycles

Phosphate metabolism; 3,0%

PROTEIN FATE19.4%

Detoxif ication; 10,4% ; Glyoxylate and TCA cycles6,0%

CELL RESCUE, DEFENCE AND VIRULENCE 10.4%

32


How is the yeast protein response?

WHAT IS THE BIOLOGICAL MEANING OF THESE RESULTS?

C. albicans inside macrophages is in a “

5

10

XPR

ESSE

D

NS

C-COMPOUND (20.3%)

PROTEIN FATE (17.5%)

“hostile environment”:- low nutrients- antimicrobial agents- low pH

0

5

OVE

R-E

XDR

OF

PRO

TEI C COMPOUND (20.3%)

METABOLISM

-5

ER-E

XPR

ESSE

D

NU

MB

ER

Lipid & fatty ac. β oxidationGlyoxylate & TCA cycles (13.5%)

Oxidative stress & detoxification (11%)

-15

-10

UN

DE REFLECTING:

- yeast shift to a starvation mode- utilization of alternative carbon sources

EXPLAINING the yeast response to such an aggressive situation.

Amino acid metabolismNucleotide metabolism

C-compound metabolism

Protein folding

Protein degradation

Assembly of protein complexes

Actin cytoskeletonCell wall

Mitochondria

Lipid & fatty ac. β oxidation

Metab. of vitamins & cofactors

Phosphate metabolismGlyoxylate & TCA cycles

Cellular transport

Oxidative stress & detoxification

Unknown

A COMPARATIVE ANALYSIS OF THE EXPRESSION PROFILE (Nº OF UP- AND DOWN-REGULATED GENES) FROM BOTH TIME POINTS


UP- AND DOWN-REGULATED GENES) FROM BOTH TIME POINTS ANALYZED

-239 regulated genes:

Transport Cell rescue

239 regulated genes:-120 and 119 differentially regulated (1.75 times induced or repressed) after 1,5 and 3 h of interaction respectively

Transcription Protein Synthesis

Transport Cell rescue

Metal ion homeostasis

Transcription Protein Synthesis

Cell rescueMetal ion homeostasis

Uncharacterized genesMetabolism and EnergyCell cycle & DNA processing

Protein FateUncharacterized genes

Metabolism and EnergyCell cycle & DNA processing

Protein FateProtein with binding function

Uncharacterized genesCell cycle & DNA processing Protein with binding function

GENETIC NETWORK ANALYSES OF UNKNOWN GENESCANDIDA HOST INTERACTION

-Osprey v1.2.0 software platform was usedplatform was usedfor visualization and manipulation of complex interaction networks

Input gene list S. cerevisiaeorthologsorthologs of unknown C. albicans genes(induced)

Biogenesis andcell organization (cell wall and nuclear pore)

Damage and DNA repair

Protein biosinthesis

Autophagy


GENETIC NETWORK ANALYSES OF UNKNOWN GENES

OSPREYOSPREY

Input gene list of S. cerevisiaeorthologs of

kunknown C. albicans genes

(REPRESSED)

Metabolism

Biogenesis and cell i tiorganization

(mitochondria and actin cytoeskeleton)

Transport

Unknown function

ACTIN CYTOSKELETON, cAMP, MITOCHONDRIA, APOPTOSIS AND AUTOPHAGY


2.79 0

1 13 2 2 3

ARP3*LSB5

1.5 h 3 h DE INTERACCIÓN

AUTOPHAGY

-Reports that connect apoptosis with actin dynamics, fLSB5

RVS161YKE2LIT1ENT4PAN1CAP2

Act

cytoske

mitochondrial dysfunction and autophagy, have been recently described in the model yeast S. cerevisiae.

Th d id d t h i d t t fCAP2MSS4SLA2ARP2Cof1pTpm2p

in

eleton

…..Thus, we decided to search in our datasets for proteins/genes belonging to these biological functions in order to carry out a new interaction network analysis.

0.995 0log2(1#5h) log2(3h)

PDE1FGR38MDM34Atp7pYIM1

cAMP signalingpathway

Mit

-A total of 32 proteins/genes, enclosed in these functions, were detected:

YIM1YME1MAM33*PHB1*Ilv5pMmf1pPor1p

ochond

-SIGNIFICANT REPRESSION IN ACTIN CYTOSKELETON AND MITOCHONDRIA

CAP1MCA1BIR1LCB1IPF12676

ria A

popt

CYTOSKELETON AND MITOCHONDRIA

-UP AND DOWN RESPONSE IN APOPTOTIC AND AUTOPHAGIC GENES

log2(1#5h) log2(3h)

IPF12676IPF12606.3eoc

SUN42VPS30

osisAutophagy

-2 5 2 5

AFTER SEVERAL NETWORKS ANALYSES….A HYPOTHETICAL MODEL OF YEAST CELL DEATH AFTER MACROPHAGE INTERACTION WAS


DNA damage

Drug - induced apoptosis

Starvation

Ceramide induced –apoptosis

(de novo pathway )

2.79 0

1 13 2 2 3

ARP3*LSB5RVS161

1.5 h 3 h

c

A HYPOTHETICAL MODEL OF YEAST CELL DEATH AFTER MACROPHAGE INTERACTION WAS SUGGESTED

(fragmentation)

LCB1(SPTLC1)

TP53 mediated

t ti

AUTOPHAGY

VPS30(APG6/ BECN1)

Starvation

BAX induced cell death

RVS161YKE2LIT1ENT4PAN1CAP2MSS4

Actin

ytoskeleto

APOPTOSIS

HumanBAX

SUN42(UTH1)

Autophagic cell deathCERAMIDE

IPF12676 (YOL086W-A

/APITD1)

apoptotic pathway

BECN1)

Heat/H2O20.995 0log2(1#5h) log2(3h)

MSS4SLA2ARP2Cof1pTpm2pPDE1FGR38

on

cAMP signalingpathway

MCA1(YCA1)

ROSrelease

IPF12606.3eoc (NMA111/HTRA2)

BIR1(XIAP)

(UTH1)

CAP1(YAP1)

PKA(Stress

2 2

Chronological agingH2O2

MDM34Atp7pYIM1YME1MAM33*PHB1*Il 5

Mitochond

ATP cAMP

PDE1/2

AMP

Inappropriate actin remodeling P 1 ( )

Cytochrome c release

(YCA1) (YAP1)response)

Replicative aging(PHB1/PHB)

Ilv5pMmf1pPor1pCAP1MCA1BIR1LCB1

dria

Apopt

FGR38 (CYR1)

remodeling(stable F-actin)

Por1p (open)Δψm

(VDAC1)(SLA2, PAN1, ARP2/3, CAP2, Cof1p…)

(MDM34, Mmd1p, Atp7p, YME1, Ilv5p…)

MitochondrialNutritional stress

log2(1#5h) log2(3h)

LCB1IPF12676IPF12606.3eocSUN42VPS30

osis

Autophagy

2 5 2 5 Mitochondrialdysfunction

Scale (fold repression or induction)

-2.5 2.5

Fernández-Arenas et al., 2007. Molecular & Cellular Proteomics

ANALYSIS OF APOPTOTIC MARKERSCANDIDA HOST INTERACTION

DNA FRAGMENTATION (hallmark of late stage apoptosis)

TUNEL ASSAY (terminal deoxynucleotidyl transferase dUTP nick-end labeling) 18 hours C albicans-Macrophages interaction18 hours C.albicans-Macrophages interaction

Collaboration with Dr Mark Ramsdale

Dept Microbiology, UCM

César NombelaGl i M lGloria MoleroLucía MonteolivaAida PitarchCarolina Fernández-HaroJ A R d i C ld óJose A. Rodriguez-CalderónVital VialasClaudia Parra

P t i U it UCMProteomics Unit UCM-Parque Científico

M. Luisa HernáezD l G tiDolores GutierrezMontserrat Martinez-GomarizFelipe ClementeIgnacio Ortea

Hospital Clínico de SalamancaAntonio Jiménez

Hospital 12 de OctubreJose María Aguado

BIO 2009 07654BIO-2009-07654CAMRETICS (ISC III)GENOMA ESPAÑA

www.ucm.es/info/gyp/proteomicagyp p

scientific meeting about clinical proteomics. 2nd...

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