ANTIGENIC RELATIONSHP OF ENTEROHEMORRHAGIC ESCHERICHU COL1 HEMOLYSIN TO OTHER RTX TOMNS A Thesis Presented to The Facutty of Graduate Studies of The University of Guelph by LESLIE ANNE MACDONALD In partial fuKlment of requirements for the degree of Master of Science September, 2000 QLeslie A. MacDonald, 2000 National Library Bibliothèque nationale 1+1 of,.,, du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Welhgton Street 395, nie Wellingt~n Ottawa ON K1A ON4 -ON KlAW Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowiog the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seli reproduire, prêter, disüibuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels rnay be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT ANTIGENIC RELATIONSHIP OF ENTEROHEMORRHAGIC ESCHERCHIA CCOLI HEMOLYSIN TO OTHER RTX TOXINS Leslie Anne MacDonald, BSc. Advisors: Dr. R. P. Johnson University of Guelph, 2000 Professor C. L. Gyles The objective of this study was to examine the antigenic relatedness of enterohemorrhagic Escherichia coli hemolysin (Ehx) to other RTX toxins, and to investigate unique arid shared Ehx epitopes. Monoclonal antibodies (Mabs) and rabbit antisera to Ehx and related toxins were created by immunizing rabbits and mice with Ehx, alpha hemolysin (Hly), or a his-tagged fiagrnent of Ehx (Ehxl-his). Toxins from recombinant and authentic Ehx-producing E. coli, Hly-producing E. coli, Mannheimia spp. and Actinobacillus spp. were exarnined for reactivity with the antibodies by Western irnmunoblotting and a neutralization test. Eight Mabs were specific for Ehx, two were specific for Hly; and others reacted with several or al1 of the RTX toxins tested. Two Mabs neutralized Ehx only, one neutraiized Hly only and a fourth neutralized Ehx, Hly and several other RTX toxins. The unique epitopes recognized by Ehx-specific Mabs were conserved among Ehx-producing E. coli. These results indicate that Ehx shares common epitopes with other RTX toxins but also possesses unique epitopes, which may be functionaily important. ACKNOWLEDGEMENTS 1 would like to express my sincm gratitude to my advisor, Dr. C. L. Gyles, for his tirne, guidance and editorial comments throughout the course of my graduate work. 1 wodd especially like to tbank my co-advisor, Dr. R. P. John, who provided on-site supervision, and offered counsel and encouragement for the duration of my shidies. This work woutd not have been possible without his support and indulgence. 1 am very grateful to the other members of my supervisory cornmittee, Dr. J. 1. Maches and Dr T. Waddell, for their guidance, suggestions and editorial advice. 1 thank the staff of the Laboratory for Foodborne Zoonoses for providing help and support and in particular, 1 wish to thank the members of the Immunology Researcti Laboratory, past and present, for providing encouragement and good humour. 1thank Amanda Mazzocco and Kim Ziebell for excellent training in molecular techniques, and SheIIey Johnson, Dawn Edmonds and Steacey Gray for preliminary work on the EHEC hemolysin. I am grateful for the moral support offered by Susan Anderson, Christine Forsberg, and Kris Rh,and for the financial support of Health Canada. 1am grateful to the staff of the Centrai Animal Facility for the humane and expert care of the Iaboratory animals used in this study. 1 thank Dr. P. Boerlin for technical assistance, advice, and use of recombinant E. cofi strains. 1am gratefd to Dr. J. S. L. Lam for making the earIy yens in the Iab a great pleasure, for nurturing my enthusiasm for my work, and for encouraging me to continue my studies. A special thank you to Tim Allman for üuly understanding, and making ou.home Iife a source of peace and constant renewal; my parents, Nice and Doug MacDonald, who have believed Ui me and my each and every endeavour al1 of my Me. 1 TABLE OF CONTENTS Page ACKNO WLEDGEMENTS ...................................................................................... i ** TABLE OF CONTENTS......................................................................................... ri... LIST OF TABLES .................................................................................................... rit LIST OF FIGURES ................................................................................................... iv 1. INTRODUCTION ........................................................... ............................1 .3 LITERATURE REViEW .......................................................................... 3 2.1 Verotoxigenic and Enterohemorrhagic Escherichia coli (EHEC) .... 3 2.2 Virulence Properties of Enterohemorrhagic Escherichia coli ........... 5 2.2.1 Shiga toxins............................................................... 6 2.2.2 Chromosomally-Encoded Virulence Factors............. 7 2.2.3 Plasmid-Encoded Potential Virulence Factors .......... 9 2.2.4 The Enterohemorrhagic Escherichia coli Hemolysin ................................................................ 11 3 . MATERlALS AND METHODS ............................................................. 31 3.1 Bacteria and Culture Methods.......................................................... 31 3.2 Antigen Preparation and Purification ................................................ 34 3.3 Production of Antisera and Antibodies.................. ... .................. 37 3.3.1 Rabbit Antisera ......................................................................37 3.3.2 Mouse Monoclonal Antibodies............................................. 38 3.4 Reactivity of Known RTX Toxins with Selected Monoclonal Antibodies......................................................................................... 45 3.4.1 Western Immunoblotting.......................................... 45 3.4.2 Neutralization Assay ................................................. 46 3.5 Epitope Specificity of Selected Monoclonal Antibodies.................. 47 3.6 Distribution of Specific Ehx Epitopes in Field Strains ..................... 49 4 . RESULTS ....................................................................................................50 4.1 Properties of Sûains.......................................................................... 50 4.2 Antigen Preparation and Purification................. +.. ..........................52 4.3 Production of Antisera and Antibodies........................................... 52 4.3.1 Rabbit Antisera ............................................................... 52 4.3.2 Mouse Monoclonal Antibodies............................................. 55 4.4 Reactivity of Known RTX Toxins with Selected Monoclonal Antibodies ........................................................................................ 55 4.5 Epitope Specificity of Selected Monoclonal Antibodies.................. 61 4.6 Distribution of Ehx-Specific Epitopes in Field IsoIates................... 65 5 . DISCUSSION.............................................................................................. 68 6 . APPENDIX ................................................................................................... 78 7 . REFERENCES............................................................................................. 82 LIST OF TABLES Table Page 2.1 The RTX family of pore-forming toxins..................................................... 15 2.2 Other proteins containing RTX motifs....................................................... 17 3.1 Bacterial strains used in this study............................................................... 32 3.2 Mouse imrnunization schedules for monoclonal antibody production ........ 39 4.1 Reactivity of selected monoclonal antibodies hmfive fisions tested by direct ELISA against Eh, Eh'-his and Hly....................................... 56 4.2 Reactivity of RTX toxins fiom various bacteria with selected monoclonal. antibodies in Western immunoblotting and neutralization assay...................................................................................... 60 4.3 Epitope specificity of monoclooal anti-RTX antibodies as determined b y biotin-streptavidin enzyme immunoassay...................... 64 4.4 Surnmary of monoclonal antibodies demonsirathg weak or non- reciprocal competrhon*. ................................................................................. 66 4.5 Reactivity of RTX toxins hmfield strains of STEC with selected Mabs in Western Unmunoblotting and neutralization assay ....................................................................................................... 67 A. 1 Prirners used in cloning the exhA gene ....................................................... 80 LIST OF FIGURES Figure Page Hemolytic phenotypes of EHEC and alpha hemolytic Escherichia coli on washed sheep red blood ce11 agar................................ 5 1 SDS-PAGEanalysis of crude Ehx and purified Ehx'-his preparations stained with Coomassie Blue ..............................................