Substrate specificities of outer membrane proteases of the omptin family in Escherichia coli, Salmonella enterica, and Citrobacter rodentium Andrea Portt, McGill University, Montreal October 9th, 2010 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master’s in Microbiology © Andrea Portt 2010 Table of Contents LIST OF ABBREVIATIONS 3 ABSTRACT 6 ACKNOWLEDGEMENTS 7 CONTRIBUTIONS OF AUTHORS 8 LITERATURE REVIEW 9 2. OMPTINS 9 3. OMPTIN SUBSTRATES 13 A. ANTIMICROBIAL PEPTIDES 13 B. BLOOD CLOTTING PROTEINS AND EXTRACELLULAR MATRIX 14 C. COMPLEMENT 16 D. TROPOMYOSIN 17 4. EVOLUTION OF OMPTINS 17 5. OMPTIN REGULATION 20 6. OMPTINS OF PATHOGENIC ENTEROBACTEREACEAE 22 A. Y. PESTIS 22 B. S. ENTERICA 23 C. SHIGELLA 25 D. ATTACHING AND EFFACING BACTERIA 25 INTRODUCTION 28 MATERIALS AND METHODS 29 1. BACTERIAL GROWTH 29 2. BACTERIAL STRAINS AND CONSTRUCTION OF PLASMIDS 29 A. STRAINS 29 B. PLASMIDS 31 3. DISK INHIBITION ASSAYS 34 4. MINIMUM INHIBITORY CONCENTRATION DETERMINATIONS 34 5. PROTEOLYTIC CLEAVAGE OF AMPS BY CELLS EXPRESSING OMPTINS 34 6. OM DISRUPTION ASSAY WITH 1-N-PHENYLNAPHTHYLAMINE 35 7. DEGRADATION OF TROPOMYOSIN BY E. COLI AND C. RODENTIUM STRAINS EXPRESSING OMPTINS 35 8. REAL-TIME QUANTITATIVE PCR 35 9. PURIFICATION OF NATIVE CROP 35 10. EXPRESSION, PURIFICATION, AND REFOLDING OF HIS-PGTE AND HIS-CROP 36 A. EXPRESSION 36 B. PURIFICATION 36 C. REFOLDING 36 11. CLEAVAGE OF C2 FRET SUBSTRATE BY WHOLE CELLS OR PURIFIED OMPTINS 37 A. WHOLE CELLS 37 B. PURIFIED OMPTINS 37 1 RESULTS 38 1. GROWTH INHIBITION OF E. COLI AND C. RODENTIUM STRAINS EXPRESSING OMPTINS BY C18G. 38 A. DISK INHIBITION 38 B. MINIMAL INHIBITORY CONCENTRATIONS 40 2. C18G CLEAVAGE BY OMPT, OMPP, AND CROP 42 A. C. RODENTIUM 42 B. S. ENTERICA 42 3. LL-37 CLEAVAGE BY CROP 42 4. SOME OMPTINS PREVENT OM DISRUPTION BY AMPS 46 A. NPN WITHOUT AMPS 46 B. C18G 46 C. PMB 46 5. TROPOMYOSIN 50 A. E. COLI BL21(DE3) 50 B. C. RODENTIUM 52 7. RT-QPCR 55 8. OMPTIN EXPRESSION AT THE OM 55 9. PURIFICATION AND ACTIVITY OF NATIVE CROP 58 10. PURIFICATION AND ACTIVITY OF HIS-PGTE AND HIS-CROP 58 DISCUSSION 64 1. OMPT, OMPP, AND CROP CONFERRED RESISTANCE TO C18G IN DISK INHIBITION AND MIC ASSAYS, PGTE DID NOT 64 2. OMPT, OMPP, AND CROP CLEAVE C18G, UNLIKE PGTE 65 3. SOME OMPTINS PREVENT OM DISRUPTION BY AMPS 67 4. TROPOMYOSIN 68 5. TIME COURSE OF FRET C2 SUBSTRATE CLEAVAGE 69 6. RT-QPCR 70 7. OM PREPARATIONS 70 8. PURIFICATION AND ACTIVITY OF NATIVE CROP 72 9. PURIFICATION AND ACTIVITY OF HIS-PGTE AND HIS-CROP 72 10. REFOLDED OMPTINS 73 11. FINAL CONCLUSIONS AND FUTURE DIRECTIONS 73 REFERENCES 75 ATTACHED PAPER 80 MOL. MICROBIOL 74, 98-111 (2009) 80 2 1 List of abbreviations 2 !2-AP – !2-Antiplasmin 3 A/E – Attaching and Effacing 4 AMP – Antimicrobial Peptide 5 C18G – Cysteine 18 Glycine, an 18 amino acid synthetic antimicrobial peptide 6 CFU – Colony Forming Unit 7 CHAPS – 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate, a 8 zwitterionic detergent 9 CIHR – Canadian Institutes for Health Research 10 CMI – les Concentrations Minimales Inhibitrices 11 DTT – Dithiothreitol 12 ECM – ExtraCellular Matrix 13 EDTA – Disodium ethylenediaminetetraacetate dihydrate, a chelating agent 14 EHEC – EnteroHemorrhagic E. coli 15 EPEC – EnteroPathogenic E. coli 16 HGT – Horizontal Gene Transfer 17 LB – Luria-Bertani broth 18 LD50 – Lethal Dose of a pathogen for 50 % of mice infected 19 LEE – Locus of Enterocyte Effacement pathogenicity island 3 20 LPS – Lipopolysaccharide 21 MAC – Membrane Attack Complex 22 mCRAMP – Mouse Cathelicidin-Related AntiMicrobial Peptide 23 MIC – Minimal Inhibitory Concentration 24 NPN - 1-N-PhenylNaphthylamine 25 NSERC – Natural Sciences and Engineering Research Council 26 OM – Outer Membrane 27 OMP - Outer Membrane Protein 28 PAM – les Peptides AntiMicrobiens 29 PBS – Phosophate-Buffered Saline 30 Plg – Plasminogen 31 RDEC – Rabbit Diarrhoeagenic E. coli 32 RT-qPCR – Real-Time Quantitative Polymerase Chain Reaction 33 Sarcosyl – N-Lauroyl-Sarcosine 34 SCV – Salmonella-Containing Vacuole 35 SDS-PAGE – Sodium Dodecyl Sulphate PolyAcrylamide Gel 36 T3SS – Type 3 Secretion System 37 TFPI – Tissue Factor Pathway Inhibitor 38 TSB – Tryptic Soy Broth 4 39 uPA – Urokinase Plaminogen Activator 40 UTI – Urinary Tract Infection 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 5 58 Abstract 59 Outer membrane proteins (OMPs) of the omptin family are present in some 60 Gram-negative bacteria. Omptins protect cells by cleaving antimicrobial peptides 61 (AMPs). For example, the Escherichia coli OmpT and OmpP impart resistance by 62 cleaving the AMP protamine, and PgtE of Salmonella enterica was also reported 63 to cleave AMPs. Despite identification of many in vitro substrates, few 64 physiological substrates have been tested. Our laboratory recently identified the 65 omptin CroP of Citrobacter rodentium, which cleaves the murine !-helical AMP 66 mCRAMP and the similarly !-helical synthetic AMP C18G. We examined AMPs 67 as substrates of several omptins. OmpT, OmpP, and CroP cleave the AMP C18G 68 more effectively than PgtE, leading to higher minimal inhibitory concentrations 69 (MICs). This suggests that AMP cleavage is an important function of OmpT, 70 OmpP, and CroP during infection. Based on initial results, we attempted to 71 compare and contrast specificity and catalytic activity of C18G cleavage by 72 purified CroP and PgtE. 73 74 Les protéines de la membrane externe de la famille omptin se trouvent dans 75 quelques bactéries à Gram négatif. En coupant les peptides antimicrobiens 76 (PAMs), les omptins protègent les bactéries. Par exemple, les omptins OmpT et 77 OmpP d’Escherichia coli, et l’omptin PgtE de Salmonella enterica, coupent les 78 PAMs. Malgré l’indentification de plusieurs substrats in vitro, peu de substrats 79 physiologiques ont été découverts. Dans notre laboratoire, on a identifié l’omptin 80 CroP de Citrobacter rodentium, qui coupe le PAM murin mCRAMP. On décrit ici 81 l’activité de plusieurs omptins contre les PAMs. OmpT, OmpP et CroP coupent le 82 PAM C18G plus efficacement que PgtE, ce qui mène à des concentrations 83 minimale inhibitrices (CMIs) plus haute. Ces résultats suggèrent que l’activité 84 catalytique d’OmpT, OmpP, et CroP est importante lors de l’infection. À la suite 85 des résultats préliminaires, on a tenté de comparer la spécificité et l’activité 86 catalytique contre C18G des protéases purifiées CroP et PgtE. 6 87 Acknowledgements 88 The author gratefully acknowledges her supervisor, Dr. Hervé Le Moual, for his 89 careful guidance through every stage of this work. Thanks are due to Dr. Valerie 90 Le Sage for her hard work with C. rodentium in the lab and in preparing the 91 Molecular Microbiology manuscript. Dr. Samantha Gruenheid provided guidance 92 and technical training. Dr. Benoit Cousineau offered insight as a committee 93 member. Special thanks are due to Jenny-Lee Thomassin, who produced the LL- 94 37 cleavage data in Figure 9. I would also like to recognize my labmates Charles 95 Viau for his encouragement and his wealth of biochemical knowledge, and Daniel 96 Ting for his balanced perspective in all things microbiological and philosophical. 97 Charles and Daniel also worked with Mg2+ to stabilize the outer membrane in 98 NPN assays. Under the supervision of Dr. France Daigle at the Université de 99 Montréal, Christine LePage and Sébastien Sabbagh undertook the RT-qPCR work 100 and construction of the S. enterica !pgtE mutant. Dr. James Taylor and Richard 101 Wargachuk were a consistent source of advice throughout this project. Thanks are 102 also due to my parents, Cam Portt and Rena Burkholder, as well as Allen 103 Larocque, Laura Canty, Carmen Evans, and Peter F. Woodford, author of 104 Scientific Writing for Graduate Students. 105 The Government on Ontario financially supported this work in the form of the 106 Ontario-Quebec Exchange Fellowship. The Canadian Institutes for Health 107 Research (CIHR), McGill University, and the Natural Sciences and Engineering 108 Research Council (NSERC) of Canada provided additional funding. 109 110 111 112 113 114 115 116 7 117 Contributions of Authors 118 Dr. Valerie Le Sage and our supervisor Dr. Hervé Le Moual undertook the 119 majority of the scientific direction, laboratory work, and writing of the Molecular 120 Microbiology paper attached to this thesis, see page 80. Under the supervision of 121 Dr. Samantha Gruenheid, Lei Zhu produced the C. rodentium phoPQ knockout. 122 Christine Lepage undertook RT-qPCR experiments under the supervision of Dr. 123 France Daigle. Andrea Portt produced the C. rodentium croP knockout, 124 contributed data to Figures 1c, 3a, 5a, and 6, produced Table 1, and helped edit 125 the manuscript. Charles Viau contributed "-galactosidase data for the effect of 126 AMPs on activation of PhoPQ in C. rodentium. 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 8 145 Literature review 146 1. OM proteins 147 The outer membrane (OM) of Gram-negative bacteria is the surface that interacts 148 with the environment and the eukaryotic host, and consequently many OM 149 proteins (OMPs) are crucial for bacterial virulence1. OMPs have many roles in 150 pathogenic Gram-negative bacteria. OMPs interact with the host as antigens, 151 receptors or adhesins, and proteases. For example, the pH 6 antigen, of Yersinia 152 pestis has antigenic properties, but it also promotes adhesion to human epithelial 153 cells via binding to phosphatidylcholine2. OM proteases may cleave bacterial or 154 host substrates. SepA is a Shigella OM protease of the autotransporter family. 155 Autotransporters transport and release one of their own domains, resulting in 156 secretion of the extracellular domain3. The secreted portion of SepA contributes to 157 tissue invasion4.