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Encoded Genes in the Biology and Pathogenesis of Escherichia Coli O157:H7

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Encoded Genes in the Biology and Pathogenesis of Escherichia Coli O157:H7 The Pennsylvania State University The Graduate School Department of Food Science THE COMPLEX INVOLVEMENT OF PROPHAGE AND PROPHAGE- ENCODED GENES IN THE BIOLOGY AND PATHOGENESIS OF ESCHERICHIA COLI O157:H7 A Dissertation in Food Science By Chun Chen © 2012 Chun Chen Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2012 ii The dissertation of Chun Chen was reviewed and approved* by the following: Edward G. Dudley Associate Professor of Food Science Dissertation Advisor Chair of the Committee Stephen J. Knabel Professor of Food Science Robert F. Roberts Professor of Food Science Interim Head of the Department of Food Science Kenneth C. Keiler Associate Professor of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School. iii Abstract Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen, notorious for its low infectious dose and its potential to cause serious diseases such as hemorrhagic colitis and hemolytic uremic syndrome. The cattle gastrointestinal tract is the major biological reservoir of E. coli O157:H7. Whole genome sequencing of this organism identified a total of 18 prophage and 6 prophage-like elements, which make up 16% of the chromosome. These horizontally transferred genetic elements encode most of the recognized virulence factors in E. coli O157:H7, including the phage-encoded Shiga toxin (Stx) and locus of enterocyte effacement (LEE), and the pre- and post-transcriptional regulators for the expression of these virulence genes. Meanwhile, prophage regions are hot spots for genomic rearrangement in this pathogen. Therefore, the overall goal of the present study was to understand the various contributions of these prophage to the virulence and epidemiologic character of E. coli O157:H7. Stx is responsible for the serious clinical manifestations of infections in humans, and Stx2 is a subgroup that is associated with highly pathogenic strains. It has been hypothesized that both the O157:H7 strain and the susceptibility of intestinal E. coli to its phage impacts Stx2 accumulation in vivo. I quantified the phage and Stx2 production by 13 E. coli O157:H7 strains and observed dramatic strain-to-strain variability. Transduction of the stx2-encoding phage from E. coli strain EDL933 into a non-toxin producing E. coli strain (C600) demonstrated that the host background affects phage iv and toxin production. Coincubation of E. coli O157:H7 or its lytic phage pools with E. coli C600 could increase the production of Stx2 and stx2-encoding phage particles. This effect varied with the O157:H7 strain, the stx2-encoding prophage, and the presence of the phage inducing agent ciprofloxacin. Of note, phage from two E. coli O157:H7 clade 8 strains were the most infectious of those screened, while an stx2c-encoding phage possessed limited infectivity. Additionally, one clade 8 strain designated PA2 produced limited Stx2 as a pure culture, but accumulated the highest toxin level among strains tested when coincubated with C600. These results suggest that the intestinal microflora may impact the outcome of an O157:H7 infection, and that toxin production observed in pure culture might not accurately reflect the levels produced in vivo. Sp11 and Sp12 are two tandem integrated and putatively defective prophage carried by E. coli O157:H7 strain Sakai. I identified 3 classes of deletions that occur within the Sp11-Sp12 region at a frequency of ca. 7.74 × 10-4. One deletion involved a precise excision of Sp11 and the other two spanned the junction of Sp11 and Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE. Alignment of the Sp11-Sp12 DNA sequence with its corresponding regions in other E. coli O157:H7 and O55:H7 strains suggested that homologous recombination rather than integrase- mediated excision is the mechanism behind these deletions. These observations explain in part a mechanism behind the previously reported genetic instability of this genomic region of E. coli O157:H7. Data from our group suggested that the E. coli O157:H7 human isolates and isolates belonging to O serogroups that are frequently associated with foodborne v outbreaks carry more copies of ileZ and argO than others. This observation supports the hypothesis that the lambdoid prophage-encoded ileZ-argN-argO operon has a regulatory effect on EHEC virulence. For the first time, I demonstrated promoter activity upstream of all seven ileZ-argN-argO operons in E. coli O157:H7 Sakai and the transcription of ileZ and argO independent of the phage late promoter. I also determined that ileZ2 encodes a functional tRNA and suggested that argN is functionally ‘redundant’. Unexpectedly, inactivation of the ileZ2-argN2-argO2 operon did not affect the production of Stx2 and the stx2-encoding phage in Sakai and phage-converted C600. These observations do not support the widespread belief that ileZ-argN-argO are required for efficient production of Stx2 and phage particles. Streptomycin binds to the ribosome of bacteria and can inhibit translation by promoting misreading of mRNA. Restrictive streptomycin-resistant (Strr) mutations increase the accuracy of the decoding process; while concomitantly decrease the rate of translation in many cases. Streptomycin is commonly used to repress the colonization resistance from intestinal microflora during investigation of the colonization and pathogenesis of E. coli O157:H7 in animal models. LEE-encoded EspA and EspB play important roles in attachment to intestinal epithelia and colonization by E. coli O157:H7. I observed a striking decrease in the secretion levels of EspA and EspB with E. coli O157:H7 Strr restrictive mutants, which carry K42T or K42I mutations in the S12 protein. However, strains carrying mutations previously defined as mildly restrictive (K87R) and non-restrictive (K42R) showed slight and indistinguishable changes in EspA and EspB secretion, respectively. Therefore, I propose that restrictive Strr mutations alter the EspA vi and EspB secretion by the bacteria and results in reduced capacity in colonizing and cause human diseases. vii TABLE OF CONTENTS List of Figures………………………………………………………………………………………………………………xiii List of Tables……………………………………………..……………………………………………………………….xvii List of Abbreviations……………………………………………………………………………………………..….…xix Acknowledgements……………………………………………………………………………………………………xxi Chapter One. Statement of the problems…………….....................................….…………………1 Chapter Two. Literature Review……………….……………………………………………………………………3 2.1 Escherichia coli O157:H7………………….….………………………............….………………..4 2.1.1 An Overview of enterohemorrhagic E. coli….....................….….……….4 2.1.2 Clinical features of E. coli O157:H7…………….....................….………….5 2.1.3 Epidemiology of E. coli O157:H7……………...................…...….……………6 2.1.4 Transmission of E. coli O157:H7…………...........….…...........………………8 2.1.5 Subtyping methods for E. coli O157:H7……...........….…...........………..9 2.1.5.1 Pulse-field gel electrophoresis…………...........…............…..10 2.1.5.2 Octamer-based genome scanning and Lineage-specific polymorphism assay…............….…...........….…...........….10 viii 2.1.5.3 Clade classification system………...........….….........…………..11 2.1.6 Pathogenesis and virulence factors of E. coli O157:H7…………….……12 2.1.6.1 The Locus for Enterocyte Effacement…...........….…...........13 2.1.6.2 Shiga toxins……………………………………….…...........….….........16 2.1.6.3 p O157…………………………………………………...........….…..........17 2.2 Bacteriophage in E. coli O157:H7…………………………………...........….…...........….18 2.2.1 An overview of bacteriophage………………………..............….…...........18 2.2.2 stx-encoding pha ge…………………………………………...........….…...........21 2.2.2.1 VT2-Sakai: an example for stx2-encoding phage….………..22 2.2.2.2 The role of stx-encoding phage in the pathogenesis of E. coli O157:H7…………...........….…...........….…...........….….......25 2.2.2.3 Quantification for stx-encoding phage............................. 26 2.2.2.3.1 Pl aque assay……………………………............….…......26 2.2.2.3.2 E lectronic microscopy………………...........….…......27 2.2.2.3.3 Quantitative PCR………………………...........….…......27 2.2.3 Phage in the phylogeny of E. coli O157:H7………...........….…...........28 2.2.3.1 The evolution of E. coli O157:H7…………………….….….........28 ix 2.2.3.2 Prophage regions as hot spots for genomic Rearrangements…………………………………………………………….30 2.3 R are c odon t RNAs……………………………...........….…...........….…………………………33 2.3.1 An overview of codon bias…………...........….…...........……………………33 2.3.2 Rare codon and bacterial virulence….............….…..........................34 2.3.2.1 The influence of leuX on the virulence of uropathogenic E. coli……...........….…...........….…...........….…................….….....37 2.3.2.2 The influence of fimU on the virulence of S. enterica serovar Typhimurium……................….…...........….….....37 2.3.3 ileZ-argN-argO tRNA gene operons………………..............….…...........38 2.4 Using streptomycin in E. coli O157:H7 animal study…..............….…...........….42 2.4.1 A b rief review of prokaryotic translation………..............….…...........42 2.4.2 S treptomycin a nd s treptomycin-resistance…….............….…...........43 2.4.2.1 An introduction to streptomycin…….….............….…........43 2.4.2.2 Different streptomycin-resistant mutations in E. coli….45 2.4.3 Application of Strr E. coli O157:H7 in animal infection study…………47 2.5 C onclusion…………………………………...........….…...........….………………………………..50
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