Colonization of cattle by non-O157 Shiga Toxin-producing Escherichia coli serotypes A Thesis submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Veterinary Microbiology In the College of Graduate Studies and Research University of Saskatchewan Saskatoon, Saskatchewan By David Jose Asper © Copyright David Jose Asper, October 2009. All rights reserved PERMISSION TO USE POSTGRADUATE THESIS In presenting this thesis in partial fulfillment of the requirements for a post graduate degree from the University of Saskatchewan, the author agrees that the libraries of this University may make it freely available for inspection. The author further agrees that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the following: Dr. Andrew A. Potter, PhD Vaccine and Infectious Disease Organization (VIDO) In his absence, permission may be granted from the head of the department of the Veterinary Microbiology or Dean of the Western College of Veterinary Medicine. It is understood that any copying, publication, or use of this thesis or part of it for financial gain shall not be allowed without the author’s written permission. It is also understood that due recognition shall be given to the author and to the University of Saskatchewan in any scholarly use which may be made of any material in this thesis. Requests for permission to copy or to make other use of materials in this thesis in whole or in part should be addressed to: Head of the Department of Veterinary Microbiology Western College of Veterinary Medicine University of Saskatchewan 52 Campus Drive Saskatoon, Saskatchewan S7N5B4 i ABSTRACT Shiga toxin-producing E. coli (STEC) is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a type III secretion system (T3SS), which forms attaching and effacing lesions (A/E). The necessary effectors which form A/E by manipulation of host signalling and actin nucleation are present on a pathogenicity island called the Locus of Enterocyte Effacement (LEE). It has been reported that vaccination of cattle with Type III-secreted proteins (T3SPs) from STEC O157 resulted in decreased shedding. In order to extend this to non- O157 STEC serotypes, we examined the serological cross-reactivity of T3SPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Groups of cattle were vaccinated with T3SPs produced from each of the serotypes and the magnitude and specificity of the responses were measured resulting in limited cross reactivity. Overall, results suggest that vaccination of cattle with T3SPs as a means of reducing the risk of STEC transmission to humans will induce protection that is serotype specific. To pursue the possibility of a cross-protective vaccine, we investigated the protective properties of a chimeric Tir protein against STEC serotypes. Several studies have reported that Tir is highly immunogenic and capable of producing high antibody titers. Potter and colleagues also demonstrated that the vaccination of cattle with ∆tir STEC O157 strain did not protect as well as the wildtype strain. We constructed thirty- mer peptides to the entire STEC O157 Tir protein, as well as to the intimin binding domain of the Tir protein from STEC serotype O26, O103 and O111. Using sera raised against STEC O157 and non-O157 T3SPs, we identified a number of immunogenic peptides containing epitopes unique to a particular serotype. Two different chimeric Tir proteins were constructed containing the STEC O157 Tir protein fused with six STEC non-O157 peptides with or without the Leukotoxin produced by Mannheimia haemolytica. However, the vaccination of mice with the chimeric protein did not protect against challenge with STEC O157 or STEC O111. These results suggest that to achieve ii cross protection against STEC serotypes using a recombinant protein vaccine, other immunogenic and protective antigens must also be included. In order to identify other immunogenic and cross-protective antigens we cloned and expressed the genes coding for 66 effectors and purified each as histidine-tagged proteins. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with T3SPs from four STEC serotypes, experimentally infected cattle and human sera from 6 HUS patients. A total of 20 proteins were recognized by at least one of the STEC T3SP- vaccinated rabbits using Western blots. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA and NleA were recognized by the majority of the samples tested. Overall, proteins such as Tir, EspB, EspD, NleA and EspA were highly immunogenic for both vaccinated and naturally infected subjects. Based on the above results, two different mixtures of secreted proteins (5 proteins and 9 proteins) were used to vaccinate mice and test the level of shedding following challenge with STEC O157. Overall, the cocktail vaccine containing 9 immunogenic effectors including Tir, EspB, EspD, NleA and EspA was capable of reducing shedding as effectively as the current STEC T3SPs vaccine, Econiche®. iii ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Dr. Andrew Potter not only for his friendship but also for his mentorship and patience which was shown through his steady guidance and valuable advice that has made me into a better writer and overall scientist. I believe that these were crucial building blocks which have created a solid plat form for my future. I would also like to thank my committee members, Dr. Lorne Babiuk, Dr. Stephen Sanche, Dr. Vikram Misra, and Dr. Wolfgang Koester for their commitment and constructive guidance throughout this project. I am extremely thankful to Neil Rawlyk for his technical support, friendship and many discussions throughout my time at VIDO. I would also like to thank Barry Carroll and the VIDO animal care services for their assistance throughout the many animal experiments. I must also acknowledge Dr. Brenda Allen, Tracy Prysliak, Dr. Sam Attah- Poku, Dr. Hugh Townsend, Joyce Sander and Gord Crockford for their assistance and friendship. A thank you also goes out to my fellow graduate students is particular Alexander Masic, Patrick Fries and Audrey Chu who have made this time easier. A special thank you is also reserved for Jean Potter for her help and belief in me throughout these years. Finally, I am forever grateful to my father, my mother and my brother Pedro for their unconditional support, encouragement and belief in me, which no matter how rough my days were they always found a way to pick me up and send me down the right path. This project was supported by grants from Canadian institutes of Health Research (CIHR) Natural Sciences and Engineering Research Council of Canada (NSERC), and Bioniche Health Sciences. iv DEDICATION For my grandfather Sergio, That his love for science continued through the generations…. v TABLE OF CONTENTS PERMISSION TO USE POSTGRADUATE THESIS ................................................... i ABSTRACT ....................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................ iv DEDICATION................................................................................................................... v TABLE OF CONTENTS ................................................................................................ vi LIST OF TABLES ............................................................................................................ x LIST OF FIGURES ......................................................................................................... xi ABBREVIATIONS USED ............................................................................................ xiv 1.0 LITERATURE REVIEW .......................................................................................... 1 1.1 Escherichia coli........................................................................................................ 1 1.1.1 Nomenclature ................................................................................................... 1 1.1.2 Shiga toxin-producing E. coli O157:H7 ......................................................... 2 1.1.3. Non-O157 serotypes ........................................................................................ 3 1.1.4 Human diseases ................................................................................................ 5 1.1.5 Animal reservoirs for STEC ......................................................................... 10 1.1.6 Modes of transmission ................................................................................... 14 1.2 STEC virulence factors......................................................................................... 20 1.2.1 Shiga Toxins ................................................................................................... 20 1.2.2 Haemolysin ....................................................................................................