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. / UOIT
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OOSHDU.10155/253 |
| Date | 01 August 2011 |
| Creators | Tilley, Derek |
| Contributors | Samis, John A. |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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