Proteomics approach on the identification of virulence factors of enteropathogenic and enterohemorrhagic escherichia coli (EPEC and EHEC) and further characterization of two effectors espb and nlei

PROTEOMICS APPROACH ON THE IDENTIFICATION OF VIRULENCE FACTORS OF ENTEROPATHOGENIC AND ENTEROHEMORRHAGIC ESCHERICHIA COLI (EPEC AND EHEC) AND FURTHER CHARACTERIZATION OF TWO EFFECTORS: ESPB AND NLEI BY LI MO (M. SC.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS I wish to express my heartfelt gratitude to my main supervisor, Professor Hew Choy Leong for his care and guidance. His clement character and esteemed research passion inspirit me through my study. I wish to express my heartful thanks to my co-supervisor, Associate Professor Leung Ka Yin for his supervision and advice. He had planned properly and provided many opportunities for my research. I would sincerely thank Professor Ilan Rosenshine from the Hebrew University, Israel, for his sound instruction and generous sharing of ideas and experiences. My Special thanks will also go to my PhD. Committee members. Thank you for spending so much time in reviewing my thesis and audit my presentation. I would like to take this opportunity to thank Dr. Lin Qingsong, Dr. John Foo, Ms Wang Xianhui and Ms Kho Say Tin from the Protein and Proteomics Center for their kind assistance in mass spectrometry, protein sequencing and data analysis. Further thank goes to Mr. Shashikant Joshi for his reviewing and suggestions on my thesis. My appreciation also goes to my previous and present lab members: Dr Yu Hongbing, Dr Seng Eng Khuan, Dr Srinivasa Rao, Dr Tan Yuan Peng, Dr Yamada, Dr. Li Zhengjun, Dr. Xie Haixia , Ms Tung Siew Lai, Mr. Zheng Jun, Mr. Peng Bo, Mr. Zhou Wenguang, Ms Tang Xuhua, Mr. Liu Yang, Mr. Wang Fan, Mr. Chen Liming, for their encouragement, help, assistance and company. I would also like to thank my good friends Wang Xiaoxing, Hu Yi, Qian Zuolei, Sheng Donglai, Luo Min, Tu Haitao, Sun Deying, Alan John Lowton. I appreciate their friendship and their valuable suggestions, advice and help throughout my study. My fellow labmates and other friends who have helped me one-way or another during the course of my project are also greatly appreciated. Last, but not least, I would like to thank my family members, my father, my mother and my sister, who are always standing by me to give their utmost support. Their courage and consolidation is the most powerful strength and is never separated by the distance, which company me throughout my PhD process. i TABLE OF CONTENTS ACKNOWLEDGEMENTS·····························································································i TABLE OF CONTENTS································································································ii LIST OF PUBLICATIONS RELATED TO THIS STUDY········································ix LIST OF FIGURES ········································································································ x LIST OF TABLES ·······································································································xiii LIST OF ABBREVIATIONS······················································································ xiv SUMMARY ·················································································································· xvi Chapter I. Introduction ·································································································· I.1. Escherichia coli-the opportunistic infectious pathogen ······································ I.1.1. Pathogenic E. coli ··························································································· I.1.2. Epidemiology of EPEC ·················································································· I.1.3. Epidemiology of EHEC ················································································· I.2. Pathogenesis and virulence factors of EPEC and EHEC··································· I.2.1. EPEC pathogenesis and virulence factors···················································· I.2.1.1. Attaching and effacing histopathology ·················································· I.2.1.2. Localized Adherence (LA) and Bundle Forming Pili (BFP) ·············· 10 I.2.1.3. EAF plasmids ························································································ 11 I.2.1.4. Invasion·································································································· 12 ii I.2.1.5. Flagella··································································································· 13 I.2.1.6. Serine Protease - EspC·········································································· 14 I.2.1.7. Heat-stable enterotoxin (EAST1).························································ 14 I.2.2. EHEC pathogenesis and virulence factors ················································· 15 I.2.2.1. Shiga toxin ····························································································· 15 I.2.2.2. Intestinal adherence factors ································································· 16 I.2.2.3. Invasion·································································································· 17 I.2.2.4. Flagella··································································································· 18 I.2.2.5. pO157 plasmid······················································································· 19 I.2.2.6. Serine protease ······················································································ 19 1.2.2.6.1. EspP ································································································ 19 I.2.2.6.2. StcE ································································································· 20 I.2.2.7. Heat-stable enterotoxin (EAST1).························································ 21 I.2.2.8. Iron transport.······················································································· 22 I.2.3. Type Three Secretion System (TTSS) in EPEC and EHEC ····················· 22 I.2.3.1. Pathogenicity island (PAI)···································································· 23 I.2.3.2. Components of TTSS in EPEC and EHEC ········································· 24 I.2.3.2.1. Components of TTSS basal body ·················································· 25 I.2.3.2.2. TTSS translocators········································································· 26 I.2.3.2.3. LEE encoded adhesin····································································· 27 I.2.3.2.4. LEE encoded TTSS regulators······················································ 29 I.2.3.2.5. Switches of TTSS translocators and effectors ······························ 29 I.2.3.3.6. TTSS chaperons ············································································· 30 iii I.2.3.3.7. TTSS secreted Effectors································································· 30 I.3. Objectives ············································································································ 33 Chapter II. Common materials and methods······························································ 35 II.1. Common used medium and buffer··································································· 35 II.2. Bacteria strains and plasmids··········································································· 35 II.3. Tissue culture····································································································· 35 II.4. Molecular biology techniques··········································································· 36 II.4.1. Genomic DNA isolation·············································································· 36 II.4.2. Cloning DNA fragments and transformation into E. coli cells················ 36 II.4.3. Analysis of plasmid DNA ··········································································· 37 II.4.4. Plasmid DNA isolation and purification ··················································· 37 II.4.5. DNA sequencing ························································································· 38 II.4.6. Sequence analysis ······················································································· 38 II.4.7. Southern hybridization ·············································································· 39 II.5. Protein techniques····························································································· 40 II.5.1. Preparation of ECPs from E. coli strains ················································· 40 II.5.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ································································································································ 40 II.5.3. Visualization of protein bands/spots (Coomassie blue staining and silver staining)·················································································································· 41 II.5.4. Western blotting ························································································· 42 iv Chapter III. Comparative proteomics analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli and their ······················· 44 Abstract······················································································································ 45 III.1. Introduction ····································································································· 46 III.2. Materials and methods ···················································································· 48 III.2.1. Bacterial strains and culture conditions·················································· 48 III.2.2. Proteins isolation and assay······································································ 49 III.2.3. One- and two-dimensional gel electrophoresis········································ 49 III.2.4. Tryptic in-gel digestion and MALDI-TOF MS analysis························· 50 III.3. Results and discussion ····················································································· 51 III.3.1. ECP production ························································································ 51 III.3.2. Extracellular Proteomes of EHEC and EPEC ········································ 59 III.3.3. Identification of Ler and IHF regulated proteins ··································· 63 III.3.4. Applications and conclusions ··································································· 67 Chapter IV. Characterization of EspB as a translocator and an effector and its involvement in EPEC autoaggregation········································································ 68 Abstract······················································································································ 69 IV.1 Introduction ······································································································ 70 IV.2. Materials and methods ···················································································· 72 IV.2.1. Bacterial strains and plasmids ································································· 72 IV.2.1. Fractionation of infected HeLa cells ························································ 73 IV.2.2. Invasion assay···························································································· 73 v IV.2.3. Examination of bacterial surface exposed structure by transmission electron microscopy (TEM) ·················································································· 74 IV.2.4. Phase contrast microscopy ······································································· 74 IV.2.5. Construction of plasmid for expression of EspB-TEM and β-lactamase based translocation assay······················································································ 75 IV.2.6. Edman N-terminal sequencing································································· 75 IV.3. Results··············································································································· 76 IV.3.1. Survey of the two forms of EspB······························································ 76 IV.3.2. Mutation of espB did not affect the secretion of other extracellular proteins··················································································································· 77 IV.3.3. Mutation of espB mutant abolished the translocation of effectors ········ 80 IV.3.4. EspB is translocated into infected HeLa cells·········································· 80 IV.3.5. EspB is involved in the autoaggregation·················································· 83 IV.3.6. EspB mutant does not form the autoaggregation ··································· 83 IV.3.7. ∆espB mutant showed less extracellular filamentous appendages········· 86 IV.3.8. ∆espB mutant has lower invasion ability ················································· 87 IV. 4. Discussion ········································································································ 89 IV. 5. Conclusion ······································································································· 92 Chapter V. Identification and Characterization of NleI, a New Non-LEE-encoded Effector of Enteropathogenic Escherichia coli (EPEC) ·············································· 93 Abstract······················································································································ 94 V.1. Introduction ······································································································· 95 vi V.2. Material and methods ······················································································· 98 V.2.1. Bacteria strains and culture conditions····················································· 98 V.2.2. Tissue culture conditions············································································ 98 V.2.3. Construction of deletion mutants and plasmids ······································· 98 V.2.4. Flow cytometric analysis ············································································ 99 V.2.5. 2-D SDS-PAGE and proteomics ······························································ 100 V.2.6. Fractionation of infected HeLa cells························································ 100 V.2.7. Expression and Immunoblot analysis······················································ 101 V.2.8. Translocation assay ·················································································· 102 V.2.9. Fluorescence microscopy for observation of translocation, transfection and actin condensation························································································ 102 V.3. Results ·············································································································· 106 V.3.1. Identification of NleI from EPEC sepL and sepD mutants ···················· 106 V.3.2. NleI is located within a prophage-associated island in EPEC ··············· 107 V.3.3. NleI is a secreted protein and the secretion of NleI is TTSS-dependent113 V.3.4. NleI is translocated into the host cells ····················································· 116 V.3.5. CesT is involved in the translocation but not the stabilization of NleI · 119 V.3.6. NleI is localized in the host cytoplasm and membrane ·························· 119 V.3.7. NleI is regulated by SepD but not regulated by Ler and SepL at the transcriptional level····························································································· 124 V.3.8. NleI is not involved in the filopodia and pedestal formation ················· 125 V.4. Discussion········································································································· 128 V.5. Conclusion········································································································ 130 vii Chapter VI. General conclusions and future directions ··········································· 132 VI.1. General conclusions ······················································································· 132 VI.2. Future directions···························································································· 134 Reference ····················································································································· 137 viii LIST OF PUBLICATIONS RELATED TO THIS STUDY 1. Li, M., I. Rosenshine, S.L. Tung, X.H. Wang, D. Friedberg, C.L. Hew, and K.Y. Leung. 2004. Comparative proteomics analysis of extracellular proteins of enterohemorrhagic and enteropathogenic Escherichia coli and their ihf and ler mutants. Appl. Environ. Microbiol. 70:5274-5282. 2. Li, M., I. Rosenshine, H.B. Yu, C. Nadler, E. Mills, C.L. Hew, and K.Y. Leung. Identification and characterization of NleI, a new non-LEE-encoded effector of enteropathogenic Escherichia coli (EPEC). (Microbes and Infection. 2006. Accepted.) 3. Li, M., I. Rosenshine, and K. Y. Leung. Characterization of EspB as a translocator and an effector and its involvement in EPEC autoaggregation. (In preparation) ix 96. Hamada, D., T. Kato, T. Ikegami, K. N. Suzuki, M. Hayashi, Y. Murooka, T. Honda, and I. Yanagihara. 2005. EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein. FEBS J. 272:756-768. 97. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580. 98. Hantke, K., Nicholson, G., Rabsch, W., and Winkelmann, G. 2003. Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN. Proc. Natl. Acad. Sci. USA 100: 3677-3682. 99. Hasman, H., T. Chakraborty, and P. Klemm. 1999. Antigen-43-mediated autoaggregation of Escherichia coli is blocked by fimbriation. J. Bacteriol. 181:4834-4841. 100. Hayashi, T., K. Makino, M. Ohnishi, K. Kurokawa, K. Ishii, K. Yokoyama, C. G. Han, E. Ohtsubo, K. Nakayama, T. Murata, M. Tanaka, T. Tobe, T. Iida, H. Takami, T. Honda, C. Sasakawa, N. Ogasawara, T. Yasunaga, S. Kuhara, T. Shiba, M. Hattori, and H. Shinagawa. 2001. Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res. 8:11-22. 101. Heath, P. T., N. K. Nik. Yusoff, and C. J. Baker. 2003. Neonatal meningitis Archives of Disease in Childhood Fetal and Neonatal Edition. 88:173-178. 102. Hedberg, C. W., S. J. Savarino, J.M. Besser, C. J. Paulus, V. M. Thelen, L. J. Myers, D. N. Cameron, T. J. Barrett, L. B. Kaper, and M. T. Osterholm. 1997. An outbreak of foodborne illness caused by Escherichia coli O39:NM, an agent not fitting into the existing scheme for classifying diarrheogenic E. coli. J. Infect. Dis. 176:1625-1628. 103. Hill, S. M., A. D. Phillips, and J. A. Walker-Smith. 1991. 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Immun. 62:41534159. 162 [...]... attaching and effacing (A/E) lesions on the epithelial cells EPEC and EHEC utilize the type III secretion systems (TTSSs) encoded on the locus of enterocyte effacement (LEE) to secrete and translocate several effectors into host cells The effectors may subvert the host signaling transduction pathways and consequently cause diseases To further understand the pathogenesis and dissect the virulence of EPEC and. .. the diagnostics and therapeutics for these pathogens Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are two of the most common human pathogens which constitute a significant risk to human health worldwide The present study attempts to investigate and understand the pathogenesis of EPEC and EHEC Approaches for understanding the pathogenesis of EPEC and EHEC, which... infection This study has established a proteomics platform for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnosis of these pathogens The discovery of new secreted proteins and new effectors by comparative proteomics approach has provided valuable information on new virulence factors in EPEC and EHEC These results further supported the notion that... pathogenic E coli and there have been significant advances in our understanding of the pathogenesis of E coli infection Urinary tract infections are a serious health problem affecting millions of people each year Most infections of the urinary tract are caused by one type of bacteria called 2 uropathogenic E coli (UPEC) and are by far the second most common type of bacteria infection in the body (Foxman... proteins and phage related proteins These novel proteins provided new candidates for exploiting potential virulence factors of EPEC and EHEC One of the major secreted proteins, EspB, was found to have two dominant forms in extracellular proteomics profiles of EPEC and EHEC The two forms of EspB were shown to remain unmodified at their N-terminus and they were unphosphorylated With a translocation signal... type strain and the espB mutant 79 Fig IV.4 Western blot analysis of Triton X-100 insoluble and soluble fractions of HeLa cells after infected with the EPEC wild type and the espB mutant and detected with anti-Tir antibody 79 Fig IV.5 Demonstration of the translocation of EPEC EspB proteins into live HeLa cells by using TEM-1 fusions and fluorescence microscopy 82 Fig IV.6 Autoaggregation of EPEC in... and on the EAF plasmid PerA is homologue to an AraC family of bacteria regulators and activate the expression of the down stream gene perC The production of PerC can increase the expression of the chromosomal eae (E coli attaching 11 and effacing) and espB (E coli secreted protein B) genes, as well as that of genes encoding 20 kD, 33 kD and 50 kD outer membrane proteins (Gomez-Duarte et al., 1995) The. .. utilizes the hemoglobin and heme released from lysed erythrocytes (Hantke et al., 2003) These virulence factors function jointly causing the infection and inflammatory symptoms in the urinary tract Meningitis is the inflammation of the membranes covering the brain and spinal cord Though multiple organisms may cause meningitis, E coli is the leading agent responsible for around 20% of the neonatal meningitis... 1990) These pedestal structures can extend up to 10 mm out from the epithelial cell in pseudopod like structures (Moon et al., 1983) It is observed that the composition of the A/E lesion contained high concentrations of polymerized filamentous actin (Knutton et al., 1989) This observation led to the development of the fluorescent-actin staining (FAS) test which enabled the screening of clones and mutants,... lesion observed in the ileum after oral inoculation of gnotobiotic piglets Note the intimate attachment of the bacteria to the enterocyte membrane with disruption of the apical cytoskeleton Note the loss of microvilli and the formation of a cup-like pedestal to which the bacterium is intimately attached (highlighted by circle) Reprinted from Baldini et al., 1983b 9 Fig I.2 Genetic organization of the . PROTEOMICS APPROACH ON THE IDENTIFICATION OF VIRULENCE FACTORS OF ENTEROPATHOGENIC AND ENTEROHEMORRHAGIC ESCHERICHIA COLI (EPEC AND EHEC) AND FURTHER CHARACTERIZATION OF TWO EFFECTORS: ESPB. understand their mechanisms of infection and to develop the diagnostics and therapeutics for these pathogens. Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). information on new virulence factors in EPEC and EHEC. These results further supported the notion that EPEC and EHEC may use multiple virulence factors to exploit the host cells and many factors