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Characterisation of New PotentialVaccine Candidates against Infectionscaused by Staphylococcus aureusInaugural-Dissertation zur Erlangung des Doktorgr

2 Introduction1997; Patel et al., 1987; Peacock et al., 1999).2.2 Antibiotic resistance of S. aureusTo date antibiotics are the only effective therapy

2 Introduction2.3 Vaccination strategies against S. aureus2.3.1 Active immunisationSince first vaccination studies employing active immunisation using

2 Introductionor SdrE significantly reduced the bacterial load in infected kidneys (Stranger-Jones et al.,2006). Moreover, as observed in the same stud

2 Introductionpreparation targeting CP5 and CP8 obtained from volunteers immunised with StaphVaxTM(Altastaph; Nabi Biopharmaceuticals) failed to prove

2 Introductiontimicrobial actions mediated by antibodies. It was shown that IgM or IgG specific for Bor-relia burgdorferi surface proteins damage the s

3 Material and Methods3.1 Material3.1.1 Chemicals and enzymesChemicals were of research grade and purchased from AppliChem, Merck, Sigma-Aldrich,ROTH

3 Material and MethodsMedia / Antibiotics CompositionLB -Medium 1 % Tryptone; 0.5 % Yeast extract; 0.5 % NaCl; pH 7.0LB-Agar1 % Tryptone; 0.5 % Yeast

3 Material and MethodsBuffers and Solutions Composition10 % SDS 100 g SDS in 1000 ml A. bidest.Prehybridisation 3.5 x SSC, 0.1 % SDS, 10 mg/ml BSA in

3 Material and MethodsBuffers and Solutions CompositionCoomassie stainingStaining solution 2.5 % Serva blue R250, 45 % Ethanol, 15 % Acetic acidDe-col

3 Material and MethodsBuffers and Solutions CompositionElution buffer 0.1 M Glycine-HCl; pH 2.5Neutralisation buffer 1 M Tris-HCl; pH 9.03.1.4 Technic

Berichterstatter: Prof. Dr. Martin KrönkeProf. Dr. Jonathan C. HowardTag der mündlichen Prüfung: 12.07.20102

3 Material and MethodsDevice Specification ProviderTank-Blotter Trans-Blot cell (16x20 cm) Bio-RadTest-Tube-Rotator Model 34528 SnjidersThermomixer Com

3 Material and MethodsDesignation ManufacturerQIAprep Spin Miniprep QiagenQIAquick PCR Purification QiagenRNeasy Mini Kit QiagenSuperscript®III first st

3 Material and MethodsGene Name Sequence (5’ to 3’)qPCR ferritinferri-f CAG CAC CAA AAA TTG ACT TTT CAA Gferri-r TCT TGA CGA GCG ATT TCA GAT AAG TTA3.

3 Material and Methods3.2 Methods3.2.1 Depletion of specific IgGs from IVIGS. aureus ATCC 29213 or E coli K12 XL1blue from an overnight (ON) culture wa

3 Material and Methodsand the pellets washed once with ice-cold TE pH 8.0, and immediately subjected to RNAisolation3.2.3 RNA isolationSubsequent to w

3 Material and MethodsThe Dye-coupling reaction was stopped by addition of 35 µl 100 mM NaOAc pH 5.2. Sam-ples intended for co-hybridisation were mixe

3 Material and Methodsvs. PBS). Recommended settings for filtering criteria were applied to exclude signals fromthe dataset, which are not exceeding ba

3 Material and Methodsurea for 2-D gel electrophoresis (2-DE). Protein concentration was determined using theBio-Rad DC assay in a 1:4 dilution accord

3 Material and Methodstransformed into E. coli BL 21 for protein expression.Clones with the highest efficiency in the expression of the respective fusi

3 Material and Methodswas neutralised by addition of a sufficient amount of 1 M Tris-HCl (pH 9.0). IgG-containingfractions were pooled and, after buffe

Contents1 Abbreviations 62 Introduction 82.1 Clinical relevance and pathogenicity of Staphylococcus aureus . . . . . . . 82.2 Antibiotic resistance of

3 Material and Methods3.2.14 In vitro opsonophagocytic killing of S. aureus by human neutrophilsTo assess killing of S. aureus by neutophils, in contr

3 Material and Methods3.2.17 Statistical analysisGrowth curve results were statistically analysed by one-way ANOVA, followed by Bonfer-roni post analy

4 Results4.1 Characterisation of the bacteriostatic effect mediated by S. aureusspecific IgGs4.1.1 Human serum inhibits in vitro growth of S. aureusIt

4 Results0 60 120 180103104105106LB25% PBS25% human serum25% HI-serum*********t (min)CFU / ml0 60 120 180103104105106LB*********50% PBS50% HI-serum50%

4 Results0 60 120 180103104105106LB25 % PBS25 % IVIG****t (min)CFU / ml0 60 120 180103104105106LB50 % PBS50 % IVIG*****t (min)CFU / mlABFigure 4.2:Ef

4 Results4.1.3 Bacteriostatic effect on S. aureus is mediated by S. aureus specific IgGsTo ensure that the observed inhibitory effect mediated by IVIG

4 Resultscontrol LB dSaPBS dSaIVIG dEcIVIG IVIG 0255075100125*********120 minCFU / ml(% of control)Figure 4.4:Effect of IVIG on in vitro growth of S.

4 Results4.1.4 Gene expression profiling of S. aureus over the course of bacteriostasisTo elucidate the mechanism leading to the observed bacteriostati

4 Results39Function unknown19Inorganic compound transport & metabolism (15)12Amino acid transport & metabolism (3)11Co-enzyme metabolism (4)8N

4 ResultsTable 4.1:Differentially expressed genes related to iron uptake and metabolism in IVIG versus dSaIVIGtreated samples revealed by microarray a

Contents3.2.9 Subtractive proteome analysis (SUPRA) . . . . . . . . . . . . . . . . 273.2.10 Cloning, expression and purification of vaccine candidates

4 ResultsTo identify differentially expressed genes in IVIG treated samples at each time point (t0,t30 and t60), microarrays were hybridised in parall

4 Results31 genes9Function unknown6Carbohydrate transport & metabolism3Energy production & conversion3Translation, ribosomal structure & b

4 ResultsAll genes falling into one of these two categories, including potential regulators are listedin Table 4.2 (for all 78 genes see Table 9.3 in

4 ResultsRogers, 1980; Goel and Kapil, 2001). Thus, quantitative real time PCR (qPCR) for selectediron related genes was applied to validate the micro

4 Resultsmicroarray qPCRt0 t30 t60-4-2024fold changet0 t30 t60-4-2024fold changeA Bfurcatalaset0 t30 t60-4-2024fold changet0 t30 t60-4-2024fold change

4 ResultsIVIG vs dSaIVIG dSaIVIG vs. PBSIVIG vs. PBSt0 t30 t60-4-2024fold changet0 t30 t60-4-2024fold changeABfurcatalaset0 t30 t60-4-2024fold changet

4 Results4.2 Identification and characterisation of potential vaccine candidatesagainst S. aureus4.2.1 Subtractive proteome analysis (SUPRA) of anchorl

4 Results204320392037/2038222120362042205020532056215521612219222224252406hp2160"2158kDa310pI21304566972037/2038222120422050205320562155216122192

4 ResultsTable 4.3: Immunogenic anchorless cell wall proteins from S. aureus ATCC 29213 identified by SUPRAGI protein Spot ID Predicted function pIa)MW

4 ResultsFurthermore, the two most obvious candidates S. aureus protein A (Spa, several spotsbetween 45 and 66 kDa) and immunodominant antigen A (IsaA

Contents7 Zusammenfassung 668 Bibliography 689 Supplement 7810 Danksagung 8611 Erklärung 8712 Lebenslauf 885

4 ResultsAE. coliLysBL21-BT1 Lyspur. BT1170130100705540352515BT1anti-BT1170130100705540352515E. coliLysBL21-BT2 Lyspur. BT2BT2anti-BT21701301007055403

4 Results10010110210310480706050403020100Serum75.4 %10010110210310480706050403020100HI-Serum77.2 %10010110210310480706050403020100anti-BT147.4 %100101

4 Results0 10 20 30 40 50 60 70 80 90 10020406080100120dSaIVIGIVIG anti-BT32.5% HI-serumno ABanti-BT1anti-BT2*******************t (min)Bacterial surv

4 Resultschallenged intravenously with 3 x 107CFU of S. aureus strain ATCC 29213 one week afterthe second booster immunisation. For two weeks infected

4 Results0 5 10 15020406080100BT1BSAP= 0,0488t (days)Percent survival0 5 10 15020406080100BT2BSAP= 0,6821t (days)Percent survival0 5 10 15020406080100

4 Results0 5 10 15020406080100BT1BSAp = 0.0168t (days)Percent survival0 5 10 15020406080100BT3BSAp = 0.2787t (days)Percent survival0 5 10 150204060801

5 DiscussionS. aureus causes apart from minor skin infections severe life-threatening invasive diseaseslike pneumonia, endocarditis and sepsis in heal

5 Discussioncould be depleted by over night co-incubation of IVIG with S. aureus (dSaIVIG), thus provingthat S. aureus specific IgGs mediate the inhibi

5 Discussionthat the expression of iron related genes was altered due to dSaIVIG treatment, hence notrepresenting the mechanism underlying bacteriosta

5 Discussion5.2 Identification and characterisation of potential vaccine candidatesagainst Staphylococcus aureusThe steadily growing interest in a prot

1 Abbreviationsaa-dUTP 5-(3-Aminoallyl)-2’-deoxyuridine 5’-triphosphateACW-proteins Anchorless cell wall proteinsANOVA Analysis of varianceAPS Ammoniu

5 Discussionremains the identification of eligible candidate proteins (Projan et al., 2006). The major pre-requisite for a target protein is the expres

5 DiscussionSince ACW proteins are not traceable by bioinformatic genome screenings, we applied aproteomic approach on an ACW-protein preparation from

5 Discussionreach statistical significance.Since the previously obtained results concerning eno, oxo and hp2160 demonstrated thatthese in vitro assays

5 DiscussionBT3.Remarkably SUPRA led to the identification of 39 immunogenic proteins within the ACW-proteins of S. aureus. The results obtained to dat

6 AbstractDue to the rapid emergence of S. aureus strains resistant to multiple antibiotics and thetherewith increased mortality rates, the developmen

6 Abstractextent using dSaIVIG were identified by MALDI-TOF analysis. SUPRA led to the identifica-tion of 37 new potential vaccine candidates among ACW

7 ZusammenfassungBedingt durch die rasante Entstehung multiresistenter S. aureus (MRSA) Stämmen undder damit einhergehenden gestiegenen Mortalität, is

7 Zusammenfassungpolyvalenten Vakzine gegen S. aureus erweitern. Zu diesem Zweck wurde eine neue sub-traktive Proteom Analyse (SUPRA) von Zellwand-ass

8 BibliographyANTELMANN, H., H. YAMAMOTO, J. SEKIGUCHI, and M. HECKER, 2002. Stabilization of cellwall proteins in Bacillus subtilis: a proteomic appr

8 BibliographyBERNARDO, K., N. PAKULAT, S. FLEER, A. SCHNAITH, O. UTERMÖHLEN, O. KRUT,S. MÜLLER, and M. KRÖNKE, 2004. Subinhibitory concentrations of

1 Abbreviationshp2160 hypothetical protein similar to esterase (spot ID 2160)IEF Isoelectric focussingIgG Immunoglobulin GIgM Immunoglobulin MIPG Immo

8 BibliographyCONNOLLY, S. E., D. G. THANASSI, and J. L. BENACH, 2004. Generation of a Complement-Independent Bactericidal IgM against a Relapsing Fev

8 Bibliographycomposed of Staphylococcus aureus type 5 and type 8 capsular polysaccharides conju-gated to Pseudomonas aeruginosa exotoxin A. Infect Im

8 BibliographyGOEL, V. and A. KAPIL, 2001. Monoclonal antibodies against the iron regulated outermembrane Proteins of Acinetobacter baumannii are bact

8 Bibliographycus aureus USA 300 clone as the predominant cause of skin and soft-tissue infections.Ann Intern Med, 144(5) 309–17KLEVENS, R. M., M. A.

8 BibliographyMAIRA-LITRAN, T., A. KROPEC, D. GOLDMANN, and G. B. PIER, 2004. Biologic propertiesand vaccine potential of the staphylococcal poly-N-ac

8 BibliographyPATEL, A. H., P. NOWLAN, E. D. WEAVERS, and T. FOSTER, 1987. Virulence of proteinA-deficient and alpha-toxin-deficient mutants of Staphylo

8 BibliographyRUPP, M. E., J. HOLLEY, H. P., J. LUTZ, P. V. DICPINIGAITIS, C. W. WOODS, D. P. LEVINE,N. VENEY, and J. FOWLER, V. G., 2007. Phase II, r

8 BibliographyVERNACHIO, J. H., A. S. BAYER, B. AMES, D. BRYANT, B. D. PRATER, P. J. SYRIBEYS,E. L. GOROVITS, and J. M. PATTI, 2006. Human immunoglobu

9 SupplementTable 9.1: Differentially expressed genes in IVIG versus dSaIVIG and IVIG versus PBSCommona)Predicted function GI proteint0 t30 t60FC 1b)F

9 SupplementList of the 236 differentially expressed genes in IVIG compared to dSaIVIG treated samples (con-tinued)Commona)Predicted function GI prote

2 Introduction2.1 Clinical relevance and pathogenicity of Staphylococcus aureusStaphlococcus aureus is a facultatively anaerobic gram-positive coccus,

9 SupplementList of the 236 differentially expressed genes in IVIG compared to dSaIVIG treated samples (con-tinued)Commona)Predicted function GI prote

9 SupplementList of the 236 differentially expressed genes in IVIG compared to dSaIVIG treated samples (con-tinued)Commona)Predicted function GI prote

9 SupplementList of the 236 differentially expressed genes in IVIG compared to dSaIVIG treated samples (con-tinued)Commona)Predicted function GI prote

9 SupplementList of the 236 differentially expressed genes in IVIG compared to dSaIVIG treated samples (con-tinued)Commona)Predicted function GI prote

9 SupplementTable 9.3: List of the 78 differentially expressed genes in IVIG compared to PBS treated samplesCommona)Predicted function GI proteint0t30

9 SupplementList of the 78 differentially expressed genes in IVIG compared to PBS treated samples (continued)Common Predicted function GI proteint0t30

10 DanksagungFür die wertvollen Diskussionen und Ideen sowie die engagierte Leitung während der ge-samten Arbeit möchte ich mich bei Herrn Prof. Dr. M

11 ErklärungIch versichere, dass ich die von mir vorgelegte Dissertation selbständig angefertigt,die benutzten Quellen und Hilfsmittel vollständig ang

12 LebenslaufAngaben zur PersonName Bettina TosettiGeburtsdatum und -ort 21.02.1978, NeussFamilienstand ledigStaatsangehörigkeit deutschHochschulausbi

2 IntroductionBesides covalently linked protein adhesins, also anchorless cell wall proteins (ACW pro-teins) mediating adhesion to extracellular matri