FUNCTIONAL STUDIES OF A TYPE III AND A NOVEL SECRETION SYSTEM IN EDWARDSIELLA TARDA ZHENG JUN NATIONAL UNIVERSITY OF SINGAPORE 2006 FUNCTIONAL STUDIES OF A TYPE III AND A NOVEL SECRETION SYSTEM IN EDWARDSIELLA TARDA ZHENG JUN (B.Sc, M.Sc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 ii ACKNOWLEDEMENTS I would like to express my heartfelt gratitude to my supervisor, Associate Professor Leung Ka Yin, for his invaluable guidance, encouragement, patience, and trust throughout my study in the lab. I am grateful to him for teaching me critical thinking and writing skills. Many thanks to Associate Professor Pan Shen Quan and Associate Professor Sanjay Swarup for their helpful advice and suggestions for my research work. Special thanks to Prof. S. J. Busby, Prof. C. W. Stephan for providing plasmids for my research work. Sincere thanks to Prof. Ilan Rosenshine for his generous sharing of ideas and experiences. I also wish to thank Prof. P. Cossart for her permission to use the figure and legend in the book she edited. I am grateful to Ms Wang Xian Hui, Ms Kho Say Tin and Mr. Shashikant Joshi from the Protein and Proteomics Centre for their ready assistance in my protein work. I would like thank Ms Tung Siew Lai for her assistance in my experiments and I am grateful to Mr. Peng Bo for his help in the phage library construction. A great deal of credit goes to my labmates - Dr. Srinivasa Rao, Dr. Yamada, Mr. Li Mo, Dr Yu HongBing, Ms Yao Fei, Ms Rasvinder Kaur D/O Nund Singh, Ms Lee Hooi Chen, Dr Xie Haixia, and Mr. Smarajit Chakraborty for their care and help during my stay. I also thank Wang Xiao Wei, Li Peng, Jiang Na Xin, Alan John Lowton, Qian Zhuo Lei and other friends in the department for helping me in one way or another during the course of my project. My parents, sisters and brother have been a great source of inspiration for all through my research. My sincere respects to them. Finally, I am deeply indebted to my wife for her love, understanding and support over the years. i TABLE OF CONTENTS ACKNOWLEDEMENTS I TABLE OF CONTENTS II LIST OF PUBLICATIONS RELATED TO THIS STUDY X LIST OF FIGURES XI LIST OF ABBREVIATIONS . XIV SUMMARY XVI Chapter I. Introduction I.1 E. tarda and its infections I.1.1 Taxonomy, identification and distribution . I.1.2 E. tarda infections I.1.2.1 Infections in humans I.1.2.2 Infections in animals I.1.3 Treatment and prevention . I.2 Virulence factors of E. tarda . I.2.1 Serum and phagocyte resistance . I.2.2 Adherence and invasion of host cell . I.2.3 Toxins, enzymes and other secreted proteins . ii I.2.4 Phosphate specific transport (PST) operon I.2.5 Type III secretion system in E. tarda . I.2.6 EVP gene cluster 13 I.3 Secretion systems in gram-negative bacteria . 14 I.3.1 Type I secretion system 14 I.3.2 Type II secretion system . 15 I.3.3 Type IV secretion system . 16 I.3.4 Type V secretion system . 16 I.3.5 Type III secretion system . 17 I.3.5.1 Regulation of type III secretion system . 19 I.3.5.2 Chaperones of TTSS 25 1.3.6 A putative novel secretion system . 28 I.4 Objectives 30 Chapter II. Common materials and methods . 32 II.1 Bacterial strains, plasmids and buffers . 32 II.2 Preparation of E. tarda cultures . 32 II.3 Molecular biology techniques 34 II.3.1 Genomic DNA isolation 34 II.3.2 Genome walking and cloning 34 iii II.3.3 Cloning and transformation into E. coli cells 35 II.3.4 Analysis of plasmid DNA . 35 II.3.5 Purification of plasmid DNA 36 II.3.6 Phage Library 36 II.3.6.1 Phage Library construction 36 II.3.6.2 Plaque screening . 37 II.3.6.3 Purification of phage DNA 37 II.3.7 DNA sequencing . 38 II.3.8 Sequence analysis 39 II.3.9 Southern hybridization 39 II.3.9.1 DNA preparation 39 II.3.9.2 Probe preparation . 40 II.3.9.3 Hybridization analysis 40 II.3.9.4 Washing and visualization . 41 II.4 Protein techniques 41 II.4.1 Preparation of extracellular proteins from E. tarda 41 II.4.2 One-dimensional polyacrlamide gel electophoresis (1D-PAGE) . 42 II.4.3 Two-dimensional polyacrlamide gel electophoresis . 43 II.4.3.1 Iso-electric focusing (IEF) . 43 II.4.3.2 Second-dimensional PAGE 44 iv II.4.4 Silver staining of protein gels 44 II.5 Western blot . 45 II.6 Animal studies 45 II.6.1 Animal model and maintenance 45 II.6.2 Fifty percent median lethal dose (LD50) studies 46 II.7 Statistical Analysis . 46 Chapter III. Regulation of a Type III and a Putative Secretion System (EVP) by EsrC in E. tarda . 47 III.1 Introduction . 49 III.2 Materials and Methods 52 III.2.1 Bacterial strains and plasmids 52 III.2.2 Construction of deletion mutants and plasmids . 52 III.2.3 LD50 determinations . 57 III.2.4 Phagocyte isolation 57 III.2.5 2D-PAGE and protein identification . 58 III.2.6 RNA isolation and RT-PCR analysis . 58 III.2.7 β-galactosidase assays 58 III.3 Results . 59 III.3.1 Sequence analysis of regulators . 59 III.3.2 Role of EsrC in E. tarda virulence and protein secretion 62 v III.3.3 Functional relationship between two-component system EsrA-EsrB and EsrC . 67 III.3.4 Regulation of TTSS apparatus genes and orf29 and orf30 69 III.3.5 EsrC regulates the EVP gene cluster . 70 III.3.6 Regulation of esrA-esrB and esrC . 73 III.4 Discussion . 76 III.4.1 EsrC is a positive regulator 76 III.4.2 EsrC regulates orf29 and orf30 and EVP . 78 III.4.3 Involvements of other regulators . 80 III.4.4 Effect of high temperature . 81 Chapter IV. EscBD is a chaperone for the TTSS translocon components EseB and EseD in E. tarda . 84 IV.1 Introduction . 86 IV.2 Materials and Methods . 88 IV.2.1 Bacterial strains and plasmids . 88 IV.2.2 Construction of deletion mutants and plasmid 89 IV.2.3 1D-PAGE, 2D-PAGE and Western analysis of proteins . 92 IV.2.4 Fractionation of bacterial cells . 93 IV.2.5 β-galactosidase assays . 94 IV.2.6 Co-purification assay . 94 IV.3 Results . 95 vi IV.3.1 Orf2 is necessary for the secretion of EseB and EseD 95 IV.3.2 Orf2 has chaperone features and is not secreted into culture 96 IV.3.3 EscBD is required for the stability of both EseB and EseD in the cytoplasm . 100 IV.3.4 EscBD is not required for the transcription of eseB and eseD 102 IV.3.5 EscBD interacts with both EseB and EseD . 104 IV.3.6 EscBD does not bind to EseB and EseD concurrently 106 IV.4 Discussion . 108 Chapter V. E. tarda contains a type VI secretion system necessary for the export of proteins involved in its pathogenesis . 111 V.1 Introduction 113 V.2 Materials and Methods . 115 V.2.1 Bacterial strains and plasmids . 115 V.2.2 Yeast strain and plasmids 117 V.2.3 Library construction 117 V.2.4 Plaque screening and Lambda DNA isolation 117 V.2.5 DNA sequencing . 117 V.2.6 Mutant construction 118 V.2.7 Edman N-terminal sequencing 118 V.2.8 Antibodies generation . 119 V.2.9 Protein Assay 119 vii V.2.10 Yeast two-hybrid analysis . 119 V.2.11 Virulence in fish 121 V.2.12 β-galactosidase assays . 121 V.2.13 Southern hybridization 121 V.2.14 Bioinformatic tools . 121 V.2.15 Nucleotide accession numbers 122 V.3 Results 122 V.3.1 Sequence analysis of EVP gene cluster 122 V.3.2 Systematic mutation of individual genes of the EVP secretion system 126 V.3.3 Identification of secreted proteins of the EVP secretion system 129 V.3.4 Identification of the start codon of essA 129 V.3.5 Association of the EVP secreted proteins . 133 V.3.6 Mutations of EVP genes led EvpC and EssA accumulation inside the bacteria . 136 V.3.7 Characteristics of EvpO and its homologs 139 V.3.8 Components of the EVP secretion system interact . 142 V.3.9 C-terminus is important for the secretion of EvpC and EssA . 144 V.3.10 EssA is under the regulation of EsrC 146 V.3.11 essA contributes to the virulence of E. tarda 146 V.3.12 Distribution of EVP genes in E. tarda strains . 149 V.4 Discussion 149 viii Grimont, P. 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Microbiology 150: 2055-2068. 185 [...]... tarda infections E tarda is a ubiquitous organism with a broad host range and can cause diseases in both humans and animals I.1.2.1 Infections in humans E tarda is the only species of this genus that can infect humans and cause diseases in humans Association of E tarda with human diseases was first reported in 1969 (Jordan and Hadley, 1969) So far, at least 300 clinical cases have been reported E tarda. .. tarda cells have been observed to be surrounded by a layer of slime (Ullah and Arai, 198 3a) and this was speculated to help in bacterial adherence to host cells Clinical E tarda isolates were invasive in a HeLa cell assay (Marques et al., 1984) Janda et al (199 1a) reported that E tarda was able to penetrate and replicate in cultured HEp-2 cell Clinical isolates of E tarda induced plasma membrane ruffles... tarda infections in humans are more common in tropical and subtropical regions (Sakazaki and Murata, 1962; Kourany et al., 1977), and in persons with exposure to the aquatic environments or exotic animals including amphibians and reptiles, and conditions leading to iron overload and dietary habits like ingestion of raw fish (Janda and Abbott, 199 3a, 3 Wu et al., 1995) The diseases caused by E tarda. .. cannot utilize many sugars, and therefore the epithet tarda means inactivity E tarda can grow on tryptic soy agar medium and form small, round, raised and transparent colonies at 24-26°C (Meyer and Bullock, 1973) The biochemical characteristics of Edwardsiella are similar to Escherichia, Shigella, and Salmonella, but it is easily differentiated on the basis of a complete set of biochemical test results... sepsis (Clarridge et al., 1980), septic arthritis (Osiri et al., 1997), bacteremia (Yang and Wang, 1999) and wound infections (Vartian and Septimus, 1990; Banks, 1992; Ashford et al., 1998) E tarda was also isolated from patients with hepatobiliary diseases and immuno-competence (Janda and Abbott, 199 3a) I.1.2.2 Infections in animals Besides causing diseases in humans, E tarda is most commonly associated... freshwater environments as well as with animals that inhabit these ecosystems, such as turtles, water tortoises, fish, toads and snakes (Plumb, 1993), and it can cause Edwardsiella septicemia, a mild to severe system disease, in warmed water fish E tarda is most prevalent in channel catfish (Meyer and Bullock, 1973) and in Japanese eels (Egusa, 1976) The outbreaks of disease caused by E tarda is typically... unravel E tarda pathogenesis 11 Fig I.2 Schematic diagram of bacterial type I to V secretion systems 18 Fig III. 1 Schematic presentation of TTSS and EVP gene clusters of E tarda PPD130/91 .60 Fig III. 2 Amino acid sequence alignments of the EsrC and other members of the AraC family of transcription regulatory proteins of TTSSs 61 Fig III. 3 Proteome analysis of E tarda PPD130/91 and. .. al., 2000), and to produce toxins and enzymes such as hemolysins (Hirono et al., 1997), catalases (Srinivasa Rao et al., 2003b) and dermatotoxins (Ullah and Arai, 1983b) for disseminating infection Recently, two important virulence mechanisms that are related to secretion systems, namely, a type III secretion system (TTSS) (Tan et al., 2005) and a putative novel secretion system, EVP (E tarda virulence... extracts, and outer membrane proteins as immunogens (Song and Kou, 1979; Salati et al., 1983; Salati, 1985; Salati and Kusuda, 198 5a and 1985b; Kawai et al., 2004; Liu et al., 2005) However, no commercial vaccine has been marketed so far I.2 Virulence factors of E tarda Information regarding E tarda virulence is rather limited, although several recent studies have identified a number of virulence factors... Edwardsiella was in Japan by Sakazaki and Murata (1962) This genus includes three members, namely E ictaluri (Hawke, 1979), E hoshinae (Grimont et al., 1980) and E tarda (Ewing et al., 1965) E ictaluri can be isolated from catfish and can cause severe infections and enteric septicemia (Hawke et al., 1981), while E hoshinae has been found in water, birds, and lizards (Grimont et al., 1980) E tarda was formerly . FUNCTIONAL STUDIES OF A TYPE III AND A NOVEL SECRETION SYSTEM IN EDWARDSIELLA TARDA ZHENG JUN NATIONAL UNIVERSITY OF SINGAPORE 2006 FUNCTIONAL STUDIES OF A TYPE III. bromo-4-chloro-3-indolyl-β-D-galactopyranoside xv SUMMARY Edwardsiella tarda is an opportunistic gram-negative bacterial pathogen affecting both animals and humans. The ability of E. tarda to cause disease is. Animal studies 45 II.6.1 Animal model and maintenance 45 II.6.2 Fifty percent median lethal dose (LD 50 ) studies 46 II.7 Statistical Analysis 46 Chapter III. Regulation of a Type III and