Characterization of a Secreted Escherichia coli O86a: K61 Protease that Inactivates Human Coagulation Factor V by Derek Tilley A thesis submitted to The Faculty of Science (Applied Bioscience Program) in conformity with the requirements for the degree of Masters of Science University of Ontario Institute of Technology Oshawa, Ontario, Canada, 2011 Copyright © Derek Tilley 2011 Abstract Background: Escherichia coli (E.coli) O86a:K61 belongs to the Enteropathogenic E. coli (EPEC) group of pathogens. Acute gastroenteritis affects 2-4 billion people annually and EPEC is associated with 10-40% of hospitalized diarrhea cases globally. Coagulation Factor (F) V circulates as an inactive procofactor (Mr 330kDa) which upon thrombin activation to the active cofactor, FVa, functions in prothombinase to accelerate prothrombin to thrombin conversion by 300,000-fold. The ability of E.coli O86a:K61 to cause intestinal hemorrhage is of interest because previous research demonstrated that during E.coli O86a:K61 sepsis in baboons, a dose-dependent inactivation of FV was observed as the bacterial dose increased. These results suggested a secreted E.coli protease may have mediated this effect on FV. This research has focused on the purification, identification, and characterization of a secreted E. coli O86a:K61 protease that inactivates FV. The final partially-purified protease inactivated FV to a 250kDa product by immunoblotting, and possessed a 900-fold increase in specific activity versus FV in human plasma compared to the culture supernatant. At least 3 proteins were observed upon SDS-PAGE. Proteolytic inactivation of FV was activated by up to 500-fold with β-mercaptoethanol and 2-fold with 1M urea. The protease was heat stable retaining all of its activity versus FV after 1h at 70°C or 80°C, and partial activity (50%) at 95°C. Proteolysis of FV was blocked by 90% with alpha-1-protease inhibitor; however, the protease was resistant to 1.5 mM PMSF, and unaffected by E64, or iodoacetamide. FV is a major regulator of the coagulation process and its inactivation by the secreted E.coli protease would be expected to result in a net bleeding tendency which may contribute to the mucosal hemorrhage observed in humans with associated hemorrhagic colitis. Proteolytic inactivation of FV is predicted to result in decreased bacterial containment by host fibrin thereby increasing pathogen survival and growth. FV inactivation by the secreted E.coli protease may be part of a novel pathogenic virulence mechanism that deregulates the blood coagulation process to enhance bacterial infectivity and transmission. ii Acknowledgements First and foremost, I offer my sincerest gratitude to my supervisor, Dr. John A. Samis, who has supported me throughout my thesis with his encouragement, and guidance whilst allowing me the room to work in my own ways. This thesis would not have been possible without his support. I would like to thank the members of my committee, Dr. Ayush Kumar, and Dr. Julia Green-Johnson for their advice, support and constructive criticism. I would like to thank all my friends and colleagues of the Applied Bioscience Program at the University of Ontario Institute of Technology for providing insight during many fruitful discussions. I would like to thank my friends and family for their ongoing support and interest in my work. I would like to thank Greg Cherry for editing the manuscript. Finally, I would like to thank Jennifer Falconer for her love and support throughout my studies. iii Table of Contents Abstract.........................................................................................................................................................ii Acknowledgements......................................................................................................................................iii Table of Contents.........................................................................................................................................iv List of Tables................................................................................................................................................vi List of Figures..............................................................................................................................................vii List of Abbreviations.....................................................................................................................................ix Introduction..................................................................................................................................................1 The Coagulation System..................................................................................................................1 Regulation of the Coagulation Cascade...........................................................................................4 Factor V............................................................................................................................................5 The Coagulation System and the Innate Immune Response...........................................................8 Bacteria and the Coagulation System..............................................................................................9 Secreted Bacterial Proteases and the Coagulation System...........................................................10 Outer Membrane Vesicles.............................................................................................................11 Bacteria and Factor V.....................................................................................................................12 Study Rationale..............................................................................................................................13 Hypothesis.....................................................................................................................................14 Study Objectives............................................................................................................................14 Materials and Methods..............................................................................................................................16 Protein Quantification...................................................................................................................16 Activity Assays................................................................................................................................17 Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE).....................................20 Western Blotting............................................................................................................................22 Column Chromatography..............................................................................................................23 Standard E. coli Culture Conditions...............................................................................................24 Standard E. coli Protease Purification............................................................................................24 iv E. coli Clinical Isolate Screen..........................................................................................................25 Transmission Electron Microscopy (TEM)......................................................................................25 N-Terminal Sequencing..................................................................................................................26 Results........................................................................................................................................................27 Discussion...................................................................................................................................................81 References..................................................................................................................................................95 v List of Tables Table 1 FV Functional Activity in NHP and in 9 Patients Who Developed DIC............................................34 Table 2 Purification Table of Protein Isolation Procedure..........................................................................67 Table 3 The Effects of Detergents on FV 1-stage Activity Assay and on E. coli Protease Activity...............74 Table 4 N-Terminal Sequencing of Sephadex G-100 Gel Filtration Fractions with the Highest Protease Specific Activity...........................................................................................................................................79 vi List of Figures Figure 1 The Blood Coagulation Cascade......................................................................................................3 Figure 2 Factor V Activation and Participation in the Prothrombinase Complex.........................................7 Figure 3 Monitoring Clot Formation in NHP with Kinetic Microplate FV 1-stage Coagulation Assay.........29 Figure 4 Standard Curve of Time of Clot Formation versus Factor V Activity in NHP using FV 1-stage Microplate Assay........................................................................................................................................31 Figure 5 Effects of Concentrated Supernatants from E. coli Strains JM109, O86a: K61, and 6 Clinical Isolates on FV by Western Blot...................................................................................................................37