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The role of the non-ribosomal peptide synthetase AusAB and its product phevalin in intracellular virulence of Staphylococcus aureus Die Rolle der nicht-ribosomalen Peptidsynthetase AusAB und ihres Produktes Phevalin in der intrazellulären Virulenz von Staphylococcus aureus Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Infection and Immunity Submitted by Sebastian Blättner from München Würzburg 2016 Submitted on: ………………………………………………………… …… Office stamp Members of the Promotionskomitee: Chairperson:_Prof Dr Markus Engstler _ Primary Supervisor: Dr Martin Fraunholz _ Supervisor (Second):_PD Dr Wilma Ziebuhr _ Supervisor (Third):_Prof Dr Thomas Rudel _ Supervisor (Fourth): (If applicable) Date of Public Defence: Date of Receipt of Certificates: Table of Content Table of content Table of content .1 Summary 1.1 Abstract 1.2 Zusammenfassung Introduction 2.1 Staphylococcus aureus 2.1.1 General information 2.1.2 Prevalence of S aureus 10 2.1.2.1 Colonization 10 2.1.2.2 Epidemiology 10 2.1.2.3 S aureus strains of sequence type 8: The success of USA300 11 2.2 Virulence factors of S aureus 12 2.2.1 Toxins 12 2.2.2.1 Alpha-hemolysin (α-toxin) 12 2.2.2.2 Beta-hemolysin (β-toxin) 13 2.2.2.3 Leukocidins 14 2.2.2.4 Enterotoxins 16 2.2.2 Secreted proteases and nucleases 17 2.2.3 Complement inhibition factors 17 2.2.4 Phenol soluble modulins 18 2.2.5 Adhesion to non-organic and organic surfaces 21 2.2.6 Small molecule products of S aureus and their association with virulence 22 2.2.7 Regulation of virulence in S aureus 23 2.2.7.1 The agr quorum sensing system 23 2.2.7.2 SaeRS two-component system 26 2.2.7.3 MarR-type transcriptional regulators: The SarA protein family 27 2.2.7.4 CodY 27 2.2.7.5 Alternative sigma factor B (σB) 28 2.3 Intracellular persistence and virulence 29 2.3.1 Invasion in non-professional phagocytes 29 2.3.2 Small colony variants (SCV) 30 Table of Content 2.3.3 Intracellular cycling of S aureus 32 2.3.4 S aureus induced host cell death 33 2.4 S aureus modulation of phagosome maturation and integrity 34 2.5 Aim of this work 37 Material and Methods 38 3.1 Material 38 3.1.1 Bacterial Strains 38 3.1.2 Cell Lines 39 3.1.3 Plasmids 39 3.1.4 Oligonucleotides 40 3.1.5 Enzymes 42 3.1.6 Buffer and Media 42 3.1.7 Kits 44 3.1.8 Chemicals 44 3.1.9 Technical Equipment 47 3.1.10 Software 47 3.2 Methods 48 3.2.1 Bacterial culture methods 48 3.2.2 Genetical manipulation of bacteria 49 3.2.3 S aureus knock-out strategies 51 3.2.4 Cell culture techniques 53 3.2.5 Cell infection protocols 54 3.2.6 PMN tests 58 3.2.7 Detection of AusAB gene expression and aureusimines production 59 3.2.8 Detection of the mitochondria phenotype of phevalin 61 3.2.9 Differences in the phosphoproteome of phevalin-treated cells 62 3.2.10 Mouse infection models 62 3.2.11.1 Pulmonary lung infection 62 3.2.11.2 Muscle abscess model 64 Results 65 4.1 Generation of a transposon insertion mutant library in S aureus LAC 65 4.2 Screening for single gene mutants strains deficient in phagosomal escape using automated microscopy 67 4.3 The non-ribosomal peptide synthetase ausAB and its influence on phagosomal escape 74 Table of Content 4.3.1 Kinetics of escape in AusAB mutants 77 4.3.2 UPLC analysis of mutant strain supernatants 80 4.3.3 Growth phase dependency of aureusimine production 81 4.3.4 Aureusimines are produced by intracellular S aureus 82 4.3.5 Genetic complementation of phevalin synthesis restores phagosomal escape of an ausB mutant 83 4.4 Mutants in ausA and ausB not affect growth in broth, hemolysis but affect intracellular cytotoxicity 84 4.4.1 Mutants in ausA and ausB not show a growth defect 84 4.4.2 No differences in hemolysis between AusAB mutants and wild type 85 4.4.3 Invasion into host cells not affected by ausAB mutation 86 4.4.4 AusAB mutants are attenuated in intracellular cytotoxicity 87 4.4.5 Cell death of host cells by S aureus supernatant not influenced by phevalin 89 4.5 Addition of synthetic phevalin can restore and enhance phagosomal escape of S aureus 91 4.5.1 Phevalin associates with host cells when added extracellularly 92 4.5.2 Phevalin does not promote the phagosomal release of fixation-killed bacteria 93 4.5.3 Phevalin does not act as a classical calpain inhibitor 94 4.5.4 Phevalin does not exhibit antimicrobial properties 95 4.6 Aureusimine expression promotes survival and cytotoxicity in leukocytes 97 4.6.1 CFU in PMN are not influenced by aureusimine production 97 4.6.2 Intracellular cytotoxicity in PMN is decreased in AusAB mutants 98 4.6.3 Phevalin reduces calcium fluxes in response to FPR-receptor stimuli in PMN 99 4.6.4 Survival and cytotoxicity of aureusimine mutants are diminished in macrophages 102 4.7 The role of ausAB in in vivo mouse infection 104 4.7.1 Mouse pulmonary lung infection model 104 4.7.2 Mouse muscle abscess infection model 106 4.8 Towards identification of the host target of S aureus-produced phevalin 107 4.9 Mitochondria elongation caused by phevalin treatment 111 4.9.1 Mitochondrial ATP production is increased in phevalin treated cells 114 Discussion 116 5.1 S aureus transposon mutant screen reveals several gene products to be involved in phagosomal escape 116 5.2 The non-ribosomal peptide synthetase AusAB involved in phagosomal escape of S aureus 120 Table of Content 5.3 Aureusimine deficiency does not affect staphylococcal growth or invasion into epithelial cells but does reduce host cell death 122 5.4 Phevalin but not tyrvalin acts in phagosomal escape of S aureus 123 5.5 Phevalin production promotes S aureus intracellular survival and cytotoxicity in leukocytes 125 5.6 The S aureus NRPS influences infection outcome in a murine lung infection model 129 5.7 Host cell proteome changes after phevalin treatment indicate direct effect on host cell gene expression 131 5.8 Mitochondrial elongation by phevalin treatment 133 5.9 Conclusion and outlook 136 References 138 Appendix 169 7.1 Abbrevations 169 7.2 List of Figures 171 7.3 List of Tables 172 7.4 Supplementary informations 173 7.5 Publications and poster presentations 182 7.6 Danksagung 183 7.7 Affidavit 184 7.8 Curriculum vitae 185 Zusammenfassung Summary 1.1 Abstract Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA) Therefore S aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it Today, infections in the USA are dominated by S aureus genotypes which are classified as USA300 and USA400, respectively Strains of genotype USA300 are responsible for about 70% of the CA infections The molecular mechanisms which render S aureus such an effective pathogen are still not understood in its entirety For decades S aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue Only recently it has been shown that S aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur S aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S aureus I thereby identified a number of genes, including a nonribosomal peptide synthetase (NRPS) The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae Zusammenfassung The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria Appendix Figure 4.29 Phevalin exhibits no chemotaxis activating properties in PMN 100 Figure 4.30 Phevalin supplementation interferes with PMN activation 101 Figure 4.31 Aureusimine mutants show decreased survival in macrophages 102 Figure 4.32 An ausB mutant causes less cell death in macrophages 103 Figure 4.33 An ausB mutant shows attenuated virulence in a pneumonia lung infection model 104 Figure 4.34 CFU recovered from lungs of infected mice 48 hours post infection not differ significantly 105 Figure 4.35 Figure 4.36 Figure 4.37 Figure 4.38 CFU recovered from removed muscle tissue of infected mice 48 hours post infection not differ significantly String DB analysis of proteins found down-regulated in total abundance and regulated via phosphorylation after phevalin treatment String DB analysis of proteins found up-regulated in total abundance and regulated via phosphorylation after phevalin treatment Confocal images and object maps of mitochondria morphology in HeLa229 cells demonstrate elongation of mitochondria by phevalin 106 108 109 112 Figure 4.39 Mitochondria length and numbers are changed in phevalin treated cells 113 Figure 4.40 Respiratory capacity and ATP production increase in phevalin treated cells 115 Figure 5.1 RNAseq data of ausAB operon shows an additional distinct transcription start site in the ausB gene 122 Figure 5.2 Detection of phagosomal escape in PMN using fluorescence flow cytometry 126 Figure 5.3 Figure 5.4 Phevalin interferes with an unknown factor of the G-protein coupled signaling pathway governing the calcium release from endoplasmic calcium stores Phevalin acts positively on S aureus intracellular survival by inducing changes in the cell metabolome 128 134 7.3 List of tables Table 3.1 Bacterial strains used in this study 38 Table 3.2 Cell lines used in this study 39 Table 3.3 Plasmids used in this study 39 Table 3.4 qRT Oligonucleotides 40 Table 3.5 Oligonucleotides used in cloning 40 Table 3.6 Oligonucleotides used for detection of Tn-Insertion sites 41 Table 3.7 Oligonucleotides used in transposon mutagenesis library generation 41 Table 3.8 Enzymes used in this study 42 Table 3.9 Buffer for molecular biology and microbiological methods 42 Table 3.10 Buffer for cell culture methods 43 Table 3.11 Kits used in this study 44 Table 3.12 Chemicals used in this study 44 Table 3.13 Score sheet depicting the determination of severity of disease in infected mice 63 Table 4.1 Annotation, replicate number, mean escape rate and standard deviation of Nebraska library mutant strains strongl attenuated in phagosomal escape Nebraska insertion mutant strains tested for phagosomal escape and their respective relative escape rates 74 Host cell genes downregulated after 30min of phevalin treatment 176 Table 7.1 Table 7.2 172 173 Appendix Table 7.3 Host cell genes downregulated after 3h of phevalin treatment 176 Table 7.10 Host cell genes upregulated after 30min of phevalin treatment 177 Table 7.11 Host cell genes upregulated after 3h of phevalin treatment 178 Table 7.12 Protein phosphorylation sites regulated in HeLa cells after 30 or h incubation with phevalin 178 7.4 Supplementary information Table 7.1: Nebraska insertion mutant strains tested for phagosomal escape and their respective relative escape rates Library label NE4 NE10 NE12 NE15 NE17 NE26 NE30 NE32 NE49 NE67 NE75 NE91 NE95 NE96 NE99 NE100 NE104 NE110 NE111 NE116 NE119 NE129 NE132 NE138 NE147 NE163 NE165 NE179 NE185 NE186 NE210 NE212 NE214 NE217 Gene Description Identifier ABC transporter ATP-binding protein putative hemolysin III drug resistance transporter, EmrB/QacA subfamily transcriptional regulator, TetR family putative drug transporter coa; staphylocoagulase precursor hydrolase, haloacid dehalogenase-like family transcriptional regulator, LysR family domain protein DNA-binding response regulator, AraC family isaB; immunodominant antigen B Cap1B; capsular polysaccharide biosynthesis protein Cap1B kdpA; K+-transporting ATPase, A subunit agrB; accessory gene regulator protein B sarU; staphylococcal accessory regulator U transcriptional regulator, Fur family FtsK/SpoIIIE family protein putative lipase/esterase transcriptional regulator, GntRfamily/aminotransferase, class I putative surface protein putative sensor histidine kinase non-ribosomal peptide synthetase DNA internalization-related competence proteinComEC/Rec2 putative transcriptional regulator putative membrane protein sensor histidine kinase aur; zinc metalloproteinase aureolysin staphylococcal accessory regulator multidrug resistance protein siderophore biosynthesis protein, IucA/IucC family fnbA; fibronectin binding protein A staphylococcal accessory regulator putative lysophospholipase siderophore biosynthesis protein, IucC family" pknB; protein kinase n % escape1 Std Dev SAUSA300_0309 SAUSA300_2129 SAUSA300_2126 SAUSA300_2509 SAUSA300_1705 SAUSA300_0224 SAUSA300_0600 SAUSA300_0095 SAUSA300_0217 SAUSA300_2573 1 1 90,2 95,2 84,6 89,6 90,4 116,8 81,4 104,9 92,4 106,0 27,4 22,1 16,6 - SAUSA300_2597 107,8 - SAUSA300_2034 SAUSA300_1989 SAUSA300_2438 SAUSA300_1448 SAUSA300_1482 SAUSA300_0641 3 113,6 15,8 88,4 125,4 105,2 78.2 1,4 18,9 5,4 29.1 SAUSA300_0110 102,1 - SAUSA300_0883 SAUSA300_1219 SAUSA300_0181 1 110,8 116,6 47,3 12,4 SAUSA300_1547 100,5 - SAUSA300_2640 SAUSA300_0275 SAUSA300_0254 SAUSA300_2572 SAUSA300_0114 SAUSA300_2360 SAUSA300_0122 SAUSA300_2441 SAUSA300_2247 SAUSA300_1710 SAUSA300_0123 SAUSA300_1113 1 1 2 1 110,0 97,0 111,7 88,9 86,0 92,8 81,2 84,1 99,9 96,5 89,0 80,6 34,3 21,9 26,3 - Normalised to the phagosomal escape rate of the USA300 JE2 wild type detected in the specific experiment If n >1 then a mean value of all single normalized escape rates is shown 173 Appendix NE218 NE251 NE258 NE262 NE274 NE286 NE287 NE290 NE294 NE296 NE324 NE331 NE332 NE334 NE338 NE354 NE355 NE362 NE367 NE369 NE374 NE383 NE386 NE391 NE392 NE399 NE400 NE406 NE410 NE415 NE420 NE426 NE431 NE432 NE434 NE444 NE447 NE453 NE454 NE457 NE462 NE481 NE483 NE505 NE512 NE513 NE514 NE519 NE528 NE530 NE535 NE539 NE541 NE551 NE554 NE557 NE558 sensor histidine kinase family protein polysaccharide biosynthesis protein cls; cardiolipin synthetase DNA-binding response regulator, LuxR family conserved hypothetical protein immunoglobulin G binding protein A precursor conserved hypothetical protein transferrin receptor signal transduction protein TRAP staphylococcal accessory protein X regulatory protein RecX exonuclease RexA iron transport associated domain protein Hyaluronate lyase precursor lip, triacylglycerol lipase precursor transcriptional regulator, GntR family protein transcriptional regulator, TetR family putative teichoic acid biosynthesis protein B arginine repressor guaC, guanosine monophosphate reductase feoB, ferrous iron transport protein B Putative membrane protein rot, staphylococcal accessory regulator Rot clfB, clumping factor B peptide ABC transporter, ATP-binding protein preprotein translocase, SecY protein iron compound ABC transporter, iron compoundbinding protein fhuA, errichrome transport ATP-binding protein fhuA ureC, urease, alpha subunit putative transcriptional regulator pbp3, penicillin-binding protein tatC, Sec-independent protein translocase TatC iron-dependent repressor sdrC protein phosphosugar-binding transcriptional regulator aminopeptidase PepS putative iron-sulfur cluster-binding protein sbi, IgG-binding protein SBI arcR, transcriptional regulator, Crp/Fnr family Peptide ABC transporter, permease protein putative membrane protein DNA-binding response regulator pepT, peptidase T transcriptional regulator, LysR family domain protein modC, molybdenum ABC transporter, ATP-binding protein ModC putative helicase sarT, staphylococcal accessory regulator T peptidase, M20/M25/M40 family putative permease transcriptional regulator, AraC family spsA, signal peptidase IA fadE, acyl-CoA synthetase FadE peptide ABC transporter, permease protein putative fibronectin/fibrinogen binding protein vraR, DNA-binding response regulator iron transport associated domain protein lukE, leukotoxin LukE 174 SAUSA300_0218 SAUSA300_0482 SAUSA300_2044 SAUSA300_1220 SAUSA300_1867 SAUSA300_0113 SAUSA300_1744 SAUSA300_0721 SAUSA300_1784 SAUSA300_0654 SAUSA300_1854 SAUSA300_0870 SAUSA300_1029 SAUSA300_2161 SAUSA300_2603 SAUSA300_0503 SAUSA300_2515 SAUSA300_0247 SAUSA300_2571 SAUSA300_1235 SAUSA300_2487 SAUSA300_0279 SAUSA300_1708 SAUSA300_2565 SAUSA300_0200 SAUSA300_2588 2 2 2 2 1 2 2 2 100,0 83,8 85.0 81,9 82,3 127,1 82,0 87,6 79,1 101,6 91,9 71,0 102,1 73,7 87,8 104,2 108,3 100,9 95,4 105,5 119,2 105,5 76,5 106,5 90,1 100,8 12,3 35.7 9,0 37,6 23,9 0,9 14,1 25,2 15,5 16,1 8,6 11,4 11,3 0,9 22,7 15,7 26,4 10,0 27,3 SAUSA300_2136 79,8 26,0 SAUSA300_0633 SAUSA300_2240 SAUSA300_2259 SAUSA300_1512 SAUSA300_0347 SAUSA300_0621 SAUSA300_0546 SAUSA300_2271 SAUSA300_1860 SAUSA300_1806 SAUSA300_2364 SAUSA300_2566 SAUSA300_0202 SAUSA300_0917 SAUSA300_0645 SAUSA300_0727 SAUSA300_0093 1 2 2 2 2 2 112,3 96,4 96,5 105,8 87,6 92,7 104,2 100,6 106,5 112,8 119,4 105,9 122,7 111,5 96,9 102,0 108,5 16,2 30,1 24,6 28,3 26,9 5,2 19,1 28,6 0,3 23,4 23,6 28,5 11,4 SAUSA300_2228 122,0 12,4 SAUSA300_2431 SAUSA300_2437 SAUSA300_1460 SAUSA300_0091 SAUSA300_2248 SAUSA300_0867 SAUSA300_0228 SAUSA300_0201 SAUSA300_1101 SAUSA300_1865 SAUSA300_1030 SAUSA300_1769 2 3 1 1 88,0 104,8 95,5 97,7 85,7 48,8 92,2 106,7 108,4 100,8 97,8 105,3 0,2 13,9 11,6 11,8 26,7 11,5 18,2 27,5 Appendix NE567 NE571 NE578 NE584 NE588 NE600 NE610 NE618 NE636 NE637 NE643 NE647 NE648 NE672 NE679 NE773 NE873 NE934 NE964 NE993 NE1065 NE1077 NE1109 NE1181 NE1188 NE1193 NE1224 NE1234 NE1240 NE1296 NE1304 NE1354 NE1363 NE1366 NE1386 NE1399 NE1449 NE1472 NE1532 NE1555 NE1607 NE1663 NE1682 NE1714 NE1756 NE1775 NE1787 NE1848 NE1868 NE1872 NE1875 NE1908 transcriptional regulator, MarR family putative transposase iron compound ABC transporter, permease protein narK, nitrite extrusion protein srrB, staphylococcal respiratory response protein, srrB putative peptidase lysP, lysine-specific permease phoR, sensory box histidine kinase PhoR exfoliative toxin A cbf1, cmp-binding-factor staphylococcal tandem lipoprotein conserved hypothetical protein glvC, PTS system, arbutin-like IIBC component homoserine kinase pbp4, penicillin-binding protein drug transporter agrC, accessory gene regulator protein C cysteine protease precursor 4'-phosphopantetheinyl transferase superfamily protein Putative hemolysin III osmoprotectant ABC transporter permease rbsK, Ribokinase rpoF, RNA polymerase sigma factor SigB putative membrane protein putative membrane protein sarA, accessory regulator A Fe/Mn family superoxide dismutase lctP, L-lactate permease lukD, leukotoxin LukD saeS, sensor histidine kinase SaeS rsp, transcription regulatory protein Alpha-hemolysin precursor srtB, sortase B catalase lukA, Leukocidin/Hemolysin toxin family protein hlgA, gamma-hemolysin component A hlgC, gamma-hemolysin component C rsbW, anti-sigma-B factor, serine-protein kinase agrA, accessory gene regulator protein A codY, transcriptional repressor CodY rsbU, sigma-B regulation protein diacylglycerol glucosyltransferase hlgB, gamma-hemolysin component B GTP pyrophosphokinase sensor histidine kinase triacylglycerol lipase srtA, sortase lukS-PV, Panton-Valentine leukocidin, LukS-PV mecA, penicillin-binding protein 2' rsbV, anti-sigma-B factor, antagonist truncated beta-hemolysin ABC transporter ATP-binding protein 175 SAUSA300_2331 SAUSA300_2263 SAUSA300_0599 SAUSA300_2333 1 98,6 87,0 77,2 122,7 28,2 46,6 SAUSA300_1441 88,4 42,5 SAUSA300_2087 SAUSA300_1628 SAUSA300_1638 SAUSA300_1065 SAUSA300_1791 SAUSA300_0102 SAUSA300_1485 SAUSA300_2270 SAUSA300_1228 SAUSA300_0629 SAUSA300_2451 SAUSA300_1991 SAUSA300_0950 2 2 2 1 96,9 89,9 92,7 99,6 115,0 110,1 95,8 73,5 113,9 100,4 79,3 13,2 85,3 10,0 5,2 17,7 8,7 15,1 2,8 8,8 11,7 SAUSA300_0182 52,4 11,7 SAUSA300_2129 SAUSA300_0707 SAUSA300_0262 SAUSA300_2022 SAUSA300_1912 SAUSA300_1912 SAUSA300_0605 SAUSA300_0135 SAUSA300_0112 SAUSA300_1768 SAUSA300_0690 SAUSA300_2326 SAUSA300_1058 SAUSA300_1034 SAUSA300_1232 SAUSA300_1974 SAUSA300_2365 SAUSA300_2366 SAUSA300_2023 SAUSA300_1992 SAUSA300_1148 SAUSA300_2025 SAUSA300_0918 SAUSA300_2367 SAUSA300_1590 SAUSA300_0646 SAUSA300_0320 SAUSA300_2467 SAUSA300_1382 SAUSA300_0032 SAUSA300_2024 SAUSA300_1918 SAUSA300_1911 1 3 5 5 4 1 2 86,7 76,5 74,7 98,3 92,6 101,9 94,9 91,2 86.6 101,2 66,0 82,3 85,3 109,6 99,9 35,9 94,0 89,4 25,6 6,4 92,2 53,3 93,3 104,0 74,7 86,5 95,9 112,3 133,8 106,2 102,5 118,3 29,3 18,1 15,0 61,5 18,0 30.9 9,9 7,9 23,1 27,1 13,7 19,2 9,2 4,4 12,2 24,8 12,3 6,5 23,9 68,1 38,1 18,3 Appendix Table 7.2: Host cell genes downregulated after 30min of phevalin treatment Gene name Protein name Norm ratio -3 Mean Proline-rich protein Dermcidin;Survival-promoting peptide;DCD-1 Apolipoprotein C-III Cornulin Coiled-coil domain-containing protein 127 Lysozyme C Centriolin ELL-associated factor Golgi apparatus membrane protein TVP23 homolog C;Golgi apparatus membrane protein TVP23 homolog B Fibronectin;Anastellin;Ugl-Y1;Ugl-Y2;Ugl-Y3 Ubiquitin carboxyl-terminal hydrolase 16 Gem-associated protein SH3 domain-binding protein RanBP-type and C3HC4-type zinc finger-containing protein RNA-binding protein 12B WD repeat domain phosphoinositide-interacting protein Meiosis arrest female protein tRNA:m(4)X modification enzyme TRM13 homolog B-cell lymphoma/leukemia 10 Protein phosphatase inhibitor 2;Protein phosphatase inhibitor 2-like protein Inverted formin-2 PRR3 DCD APOC3 CRNN CCDC127 LYZ CNTRL EAF1 NaN 0,05 0,01 NaN 0,04 0,07 0,07 0,08 0,04 0,04 0,08 0,04 0,06 0,07 0,10 NaN 0,04 NaN NaN 0,05 0,08 NaN NaN 0,33 0,0 0,0 0,0 0,0 0,1 0,1 0,1 0,2 TVP23C;TVP23B NaN 0,32 0,30 0,3 FN1 USP16 GEMIN2 SH3BP5 RBCK1 RBM12B WDR45 KIAA0430 TRMT13 BCL10 PPP1R2;PPP1R2P3 INF2 0,28 0,39 0,35 0,59 0,73 0,70 0,53 NaN 0,47 0,62 0,63 0,70 0,44 NaN 0,47 0,24 0,37 0,35 0,65 0,55 0,75 NaN 0,69 0,64 0,26 0,39 NaN NaN 0,46 0,66 0,62 0,66 0,61 0,66 0,61 0,64 0,3 0,4 0,4 0,4 0,5 0,6 0,6 0,6 0,6 0,6 0,6 0,7 Table 7.3: Host cell genes downregulated after 3h of phevalin treatment Gene name Protein name Dermcidin;Survival-promoting peptide;DCD-1 Centriolin Apolipoprotein C-III Coiled-coil domain-containing protein 127 DCD CNTRL APOC3 CCDC127 176 Norm ratio -3 0,03 0,09 0,17 0,09 0,06 0,05 0,04 NaN Mean 0,07 0,04 NaN 0,13 0,1 0,1 0,1 0,1 Appendix Antithrombin-III 60S ribosome subunit biogenesis protein NIP7 homolog Glutathione S-transferase A3 Membrane protein FAM174A Protein FAM208B Isoprenoid synthase domain-containing protein Synaptosomal-associated protein 25 Uncharacterized protein C18orf25 Interferon-induced 35 kDa protein Protein phosphatase inhibitor 2;Protein phosphatase inhibitor 2-like protein NAD(P)H dehydrogenase [quinone] Gem-associated protein Non-canonical poly(A) RNA polymerase PAPD7 Thiopurine S-methyltransferase Cytosolic endo-beta-N-acetylglucosaminidase Piezo-type mechanosensitive ion channel component E3 ubiquitin-protein ligase RNF138 SERPINC1 NIP7 GSTA3 FAM174A FAM208B ISPD SNAP25 C18orf25 IFI35 PPP1R2;PPP1R2P3 NQO1 GEMIN2 PAPD7 TPMT ENGASE PIEZO1 RNF138 0,18 0,20 0,37 NaN 0,32 NaN NaN NaN NaN 0,44 0,34 0,43 0,69 0,39 NaN 0,62 0,65 0,18 0,29 0,16 0,51 0,43 0,47 0,57 0,27 0,35 0,49 0,76 NaN 0,35 0,64 0,62 0,66 0,80 NaN 0,14 NaN 0,08 0,32 0,30 0,25 0,59 0,56 0,46 0,38 0,63 0,63 0,67 0,52 0,48 0,60 0,2 0,2 0,3 0,3 0,4 0,4 0,4 0,4 0,5 0,5 0,5 0,5 0,6 0,6 0,6 0,6 0,7 Table 7.4: Host cell genes upregulated after 30min of phevalin treatment Gene name Protein name Hemicentin-2 HEAT repeat-containing protein 5B E2F-associated phosphoprotein AP-1 complex subunit sigma-2 CDK2-associated and cullin domain-containing protein Tuberin Cytochrome c oxidase subunit 7A2, mitochondrial Nuclear receptor coactivator Integrin beta-1-binding protein R3H domain-containing protein Receptor-interacting serine/threonine-protein kinase Zinc finger CCHC-type and RNA-binding motif-containing protein HMCN2 HEATR5B EAPP AP1S2 CACUL1 TSC2 COX7A2 NCOA3 ITGB1BP1 R3HDM2 RIPK2 ZCRB1 177 Norm ratio -3 NaN 5,66 NaN NaN 2,64 3,19 2,69 1,33 1,97 1,89 1,68 1,30 35,94 NaN 6,27 5,97 1,82 1,86 1,50 2,34 NaN NaN 1,86 2,19 Mean 14,45 4,82 3,70 3,30 NaN 1,44 1,71 1,96 1,54 1,53 1,51 1,56 25,2 5,2 5,0 4,6 2,2 2,2 2,0 1,9 1,8 1,7 1,7 1,7 Appendix Table 7.5: Host cell genes upregulated after 3h of phevalin treatment Gene name Protein name BUD13 homolog Lysine-specific demethylase 3A Egl nine homolog HEAT repeat-containing protein 5B Mitochondrial calcium uniporter regulator Vacuolar protein sorting-associated protein 26B Calcium uniporter protein, mitochondrial SOSS complex subunit B2 BUD13 KDM3A EGLN1 HEATR5B MCUR1 VPS26B MCU NABP1 Norm ratio -3 7,65 6,35 2,16 NaN 1,57 1,78 1,56 NaN 3,28 1,43 NaN 2,56 1,62 NaN NaN 1,51 Mean NaN 1,92 4,19 3,68 2,99 1,63 1,81 1,53 5,5 3,2 3,2 3,1 2,1 1,7 1,7 1,5 Table 7.6: Protein phosphorylation sites regulated in HeLa cells after 30 or h incubation with phevalin Gene name Protein name Amino Acid Zinc finger protein 148 ZNF148 S Zinc finger protein 148 ZNF148 S Zinc finger protein 148 ZNF148 T LIM domain only protein LMO7 S E3 ubiquitin-protein ligase HUWE1 HUWE1 S Histone deacetylase complex subunit SAP130 SAP130 S Brain-specific angiogenesis inhibitor 1-associated protein BAIAP2 T Pre-mRNA cleavage complex protein Pcf11 PCF11 S Myotubularin-related protein MTMR3 S Pericentrin PCNT S Shootin-1 KIAA1598 S SH2B adapter protein SH2B1 S SH2B adapter protein SH2B1 S Borealin CDCA8 S Band 4.1-like protein EPB41L1 S Histone deacetylase HDAC2 S 178 Position 306 311 305 417 3539 896 360 368 633 3242 494 125 126 219 578 392 Mean Reg 30 Mean Reg h 70,97 106,40 70,97 106,40 22,64 106,40 11,62 5,83 8,24 6,68 7,88 - 7,81 2,58 7,06 - 5,66 2,20 5,57 - 5,47 - 4,39 3,34 4,39 2,45 4,33 2,46 3,68 1,37 3,64 0,95 Appendix Pre-mRNA cleavage complex protein Pcf11 PCF11 S Na(+)/H(+) exchange regulatory cofactor NHE-RF2 SLC9A3R2 S Pre-mRNA cleavage complex protein Pcf11 PCF11 S Rapamycin-insensitive companion of mTOR RICTOR S Mitogen-activated protein kinase-binding protein MAPKBP1 S Histone-lysine N-methyltransferase, H3 lysine-79 specific DOT1L S Remodeling and spacing factor RSF1 S Protein ITFG3 ITFG3 S Fanconi anemia group J protein BRIP1 S Chromatin assembly factor subunit A CHAF1A S G protein-regulated inducer of neurite outgrowth GPRIN1 S Insulin receptor substrate IRS2 S SH3 domain-binding protein 5-like SH3BP5L S tRNA (guanine-N(7)-)-methyltransferase METTL1 S Polymerase I and transcript release factor PTRF T UPF0562 protein C7orf55;Putative RNA-binding protein Luc7-like C7orf55;LUC7L2 S Beta-parvin PARVB S SH3 domain-binding protein 5-like SH3BP5L S E3 ubiquitin-protein ligase NEDD4-like NEDD4L S Ankyrin repeat and SAM domain-containing protein 1A ANKS1A S Remodeling and spacing factor RSF1 S CTD small phosphatase-like protein CTDSPL2 S Nuclear factor B-type NFIB S C2 domain-containing protein C2CD5 S Coilin COIL T Centrosomal protein of 131 kDa CEP131 T Mitogen-activated protein kinase kinase kinase MLT ZAK S Ankyrin repeat domain-containing protein 17 ANKRD17 S E3 SUMO-protein ligase NSE2 NSMCE2 S La-related protein LARP1 T Centrosomal protein of 72 kDa CEP72 S Nucleophosmin NPM1 T 179 370 20 372 21 764 1032 573 21 128 141 452 577 362 27 302 419 358 305 663 366 104 312 659 122 93 593 1939 116 858 237 219 2,93 2,87 2,91 4,09 2,71 2,87 2,42 2,19 2,40 1,88 2,35 0,79 2,32 12,72 2,16 1,01 2,07 1,44 1,99 1,08 1,98 - 1,98 - 1,90 1,47 1,83 1,98 1,79 0,68 1,77 0,89 1,76 0,83 1,65 1,76 1,61 - 1,56 - 1,51 1,50 1,47 2,64 1,36 0,43 1,29 1,33 1,29 1,40 1,22 0,45 1,11 1,68 1,04 0,54 1,01 0,44 0,99 3,91 0,91 0,42 0,89 0,44 Appendix Neuroblast differentiation-associated protein AHNAK AHNAK S Pyruvate kinase PKM PKM S Nuclear factor X-type Hematological and neurological expressed protein;Hematological and neurological expressed protein, N-terminally processed Atrophin-1 NFIX S HN1 S ATN1 S Zinc finger C3H1 domain-containing protein ZFC3H1 S Collagen alpha-1(I) chain COL1A1 S Band 4.1-like protein EPB41L1 S Vasculin GPBP1 S Arginine/serine-rich coiled-coil protein RSRC2 S Mediator of DNA damage checkpoint protein MDC1 T Hepatocyte growth factor receptor MET Y Arginine/serine-rich coiled-coil protein RSRC2 T Protein VPRBP VPRBP S Zinc finger protein 574 ZNF574 T Rho guanine nucleotide exchange factor ARHGEF2 S Spectrin beta chain, non-erythrocytic SPTBN1 S ADP-ribosylation factor GTPase-activating protein ARFGAP1 S Dedicator of cytokinesis protein DOCK5 S Rho guanine nucleotide exchange factor 16 ARHGEF16 S WD repeat-containing protein WDR7 S Rho GTPase-activating protein 21 ARHGAP21 S RING finger protein 219 RNF219 S Myosin phosphatase Rho-interacting protein MPRIP S Huntingtin HTT S N-acetyltransferase ESCO2 ESCO2 S Eukaryotic translation initiation factor 2-alpha kinase EIF2AK4 T DNA-directed RNA polymerase I subunit RPA43 TWISTNB T Transcription factor HIVEP2 HIVEP2 S Cyclin-dependent kinase 12 CDK12 S Cyclin-dependent kinase 12 CDK12 S 180 5863 97 293 80 34 655 1125 541 314 17 869 1235 16 894 306 668 14 230 1824 174 935 1095 210 977 419 244 667 322 2300 274 276 0,86 0,54 0,81 3,46 0,81 0,33 0,78 0,43 0,72 0,49 0,72 0,55 0,67 0,59 0,67 0,46 0,67 0,84 0,64 0,47 0,64 0,47 0,63 1,14 0,61 0,38 0,60 0,60 0,78 0,58 0,70 0,54 0,53 0,42 0,53 0,55 0,49 0,53 0,47 0,22 0,47 - 0,45 - 0,42 0,93 0,41 1,00 0,40 - 0,32 0,26 0,26 0,72 0,22 0,18 0,20 0,76 0,20 0,76 Appendix Nuclear receptor coactivator NCOA2 S Parathymosin PTMS S Parathymosin PTMS S Cytochrome c oxidase subunit 7A-related protein, mitochondrial COX7A2L Y Remodeling and spacing factor RSF1 S TATA-binding protein-associated factor 172 BTAF1 S Tumor necrosis factor alpha-induced protein 3;A20p50;A20p37 TNFAIP3 S Transcription factor HIVEP2 HIVEP2 S Enhancer of mRNA-decapping protein EDC4 S Protein KIBRA WWC1 S Ubiquitin carboxyl-terminal hydrolase BAP1 BAP1 S Death-inducer obliterator DIDO1 S 181 493 1251 91 641 2301 780 931 325 1312 0,19 - 0,18 0,09 0,18 0,09 0,08 0,08 0,08 0,13 - 0,09 - 0,59 - 0,14 - 2,87 - 0,51 - 0,24 - 0,45 Appendix 7.5 Publications and poster presentations Publications Blättner S., Das S., Paprotka K., Eilers U., Krischke M., Kretschmer D., Rennele CW., Dittrich M., Müller T., Schuelein-Voelk C., Hertlein T., Mueller MJ., Huettel B., Reinhardt R., Ohlsen K., Rudel T., Fraunholz MJ., (2016) Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes PLoS Pathog 12(9): e1005857 doi: 10.1371/journal.ppat.1005857 Das S, Lindemann C, Young BC, Muller J, Österreich B, Ternette N, Winkler AC, Paprotka K, Reinhardt R, Förstner KU, Allen E, Flaxman A, Yamaguchi Y, Rollier CS, van Diemen P, Blättner S, Remmele CW, Selle M, Dittrich M, Müller T, Vogel J, Ohlsen K, Crook DW, Massey R, Wilson DJ, Rudel T, Wyllie DH, Fraunholz MJ., (2016) Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation Proc Natl Acad Sci U S A 31;113(22):E3101-10 doi: 10.1073/pnas.1520255113 Poster presentations Blättner S., Das S., Eilers U., Schuelein-Voelk C., Rudel T., Fraunholz MJ., (2014) Identification of factors mediating phagolysosomal escape of Staphylococcus aureus, 3rd Mol Micro Meeting Wuerzburg Blättner S., Eilers U., Schuelein-Voelk C., Eilers M., Fraunholz MJ., (2014) Novel Staphylococcus aureus phagosomal escape factors identified by automated microscopy, Gemeinsame Konferenz von DGHM und VAAM Blättner S., Das S., Eilers U., Krischke M., Kretschmer D., Schuelein-Voelk C., Eilers M., Rudel T., Fraunholz MJ., (2015) A non-ribosomal peptide synthase is important for intracellular survival of Staphylococcus aureus, EMBL Symposium: New Approaches and Concepts in Microbiology 182 Appendix 7.6 Danksagung Meinen herzlichen Dank möchte ich ausdrücken an… …Dr Martin Fraunholz, dafür, dass er beständig an mich und an meine Arbeit geglaubt hat, dass er mir immer mit seinem Rat zur Seite stand und dafür, dass er mir dabei geholfen hat diese Thesis in ihrer jetzigen Form zu vollenden …Prof Dr Thomas Rudel für die Chance meine Doktorarbeit an diesem Lehrstuhl ablegen zu dürfen Für alle konstruktive Kritik und die Unterstützung, die ich über die Jahre hier erfahren habe …alle lieben Menschen, die mich ganz oder teilweise in meiner Zeit hier am Lehrstuhl begleitet und unterstützt haben Speziell Kerstin Paprotka möchte ich dafür danken, wie oft Sie mir geholfen hat, wenn ihre Expertise von Nöten war Anastasija Reimer gebührt ebenfalls besonderer Dank bei allen Experimenten, die wir zusammen durchgeführt haben …meine Freunden, die ich hier in Würzburg gewonnen habe und die mir die Zeit hier so versüßt haben Claudia Sibilski, Maria Kupper, Jo-Ana Herweg, Suvagata Roy Chowdhury und Sudip Das Vielen Dank, dass ihr immer da wart und für den ganzen Spaß, den wir zusammen hatten …meine Familie, die auch so manches Mal mein Jammern ertragen musste, aber die mich immer unterstützt hat, wenn ich es nötig hatte Ich könnte euch niemals alles zurückzahlen, was ihr mir bis zum heutigen Tag an Liebe und Freude mitgegeben habt …meinen ganz besonderen Schatz, Natalie, die mir immer ein Halt war, auch in unruhigeren Zeiten, in denen ich gern an mir und dem Weg, den ich beschritten hatte, zweifelte Du bist meine Sonne, die mir Kraft gibt und du bist mein Stern, der mir den Weg zeigt, wenn ich einmal nicht weiter weiß Ich liebe dich 183 Appendix 7.7 Affidavit I hereby confirm that my thesis entitled “The role of the non-ribosomal peptide synthetase and its product phevalin in intracellular virulence of Staphylococcus aureus” is the result of my own work I did not receive any help or support from commercial consultants All sources and / or materials applied are listed and specified in the thesis Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form Place, Date Signature Hiermit erkläre ich an Eides statt, die Dissertation „The role of the non-ribosomal peptide synthetase and its product phevalin in intracellular virulence of Staphylococcus aureus” eigenständig, d.h insbesondere selbstständig und ohne Hilfe eines kommerziellen Promotionsberaters, angefertigt und keine anderen als die von mir angegebenen Quellen und Hilfsmittel verwendet zu haben Ich erkläre außerdem, dass die Dissertation weder in gleicher noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat Ort, Datum Unterschrift 184 Appendix 7.8 Curriculum vitae 10/2016 – 01/2017 Berufliche Erfahrungen Referent für Projekt- und Qualitätsmanagement, Weiterbildung WBS Training AG, Landau DEKRA-Zertifizierung zum Qualitätsmanagementbeauftragten Überwachung von QM-Systemen ISO 9001:2015, ISO 13485 10/2012 – 03/2016 Wissenschaftlicher Mitarbeiter, Lehrstuhl für Mikrobiologie, Universität Würzburg Pathogen-Wirt Interaktionen in vitro / in vivo Analyse diverser Hochdurchsatz-Datensätze in den Bereichen Metabolomics, Proteomics und Phosphoproteomics Analyse von mikroskopischen Hochdurchsatz-Experimenten Zellkultur und lentivirale Manipulation humaner Zellen 01/2012 – 05/2012 Hilfswissenschaftler, Lehrstuhl für Mikrobiologie, Technische Universität Kaiserslautern Genetische Manipulation gram-positiver Bakterien Evaluierung bakterieller Antibiotika-Resistenzspektren 10/2012 - 12/2016 Akademische Ausbildung Promotionsstudent, Lehrstuhl für Mikrobiologie, Universität Würzburg Betreuung und Einarbeitung von Studenten auf Master- sowie Bachelor-Niveau Erarbeitung und Standardisierung molekular- und mikrobiologischer Experimente 04/2006 - 12/2011 Studium der Biologie, Technische Universität Kaiserslautern Abschluss Diplom (Gesamtnote 1,5) Abschlussarbeit am Lehrstuhl für Mikrobiologie (Teilnote 1,3) Abschluss Nebenfach BWL (Teilnote 1,3) 08/1996 – 03/2005 Abitur, Sickingen-Gymnasium, Landstuhl (Pfalz) 185 Appendix Sprachen Kenntnisse Englisch – Fliend Franzưsisch - Grundkenntnisse EDV MS Office Statistica Weiterbildungen Quality Management and Audit in Biotech Industries Good Clinical Practice (GCP) Neuere Methoden der Zellanalytik Patent Law in the Life Sciences Good Scientific Practice GLP - GMP Publikationen Das S, Lindemann C, Young BC, Muller J, Österreich B, Ternette N, Winkler AC, Paprotka K, Reinhardt R, Förstner KU, Allen E, Flaxman A, Yamaguchi Y, Rollier CS, van Diemen P, Blättner S, Remmele CW, Selle M, Dittrich M, Müller T, Vogel J, Ohlsen K, Crook DW, Massey R, Wilson DJ, Rudel T, Wyllie DH, Fraunholz MJ., Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation., Proc Natl Acad Sci U S A 2016 May 31;113(22):E3101-10 Blättner S, Das S, Paprotka K, Eilers U, KrischkeM, Kretschmer D, Remmele C, Dittrich M, Müller T, Schühlein-Völk C, Hertlein T, Müller MJ, Hüttel B, Reinhardt R, Ohlsen K, Rudel T, Fraunholz MJ., Staphylococcus aureus exploits a non-ribosomal cyclic dipeptide to modulate survival within epithelial cells and phagocytes., Plos Pathog 12(9): e1005857 doi: 10.1371/journal.ppat.1005857 Würzburg, den 01.12.2016 186