Characterization of OmpT: An outer membrane vesicle protease Escherichia coli that attenuates blood coagulation.

Abstract

Escherichia coli O157:H7 is an emerging food-borne and water-borne pathogen that belongs to Enterohemorrhaghic pathogenic group of bacteria. E. coli O157:H7 colonizes the gastrointestinal tract and is responsible for hemorrahagic colitis, renal failure, bloody diarrhoea, hemolytic uremic syndrome, thrombocytopenia, hemolytic anemia and other systemic problems. E. coli O157:H7 is responsible for outbreaks of Enterohemorrhaghic E. coli infections and causes significant mortality and morbidity globally. Factor V (Mr-330,00Da) is a procofactor that upon activation to FVa profoundly enhances thrombin generation and fibrin clot formation as part of prothrombinase. The fibrin clot immobolizes the pathogen and allows the immune system to target and destroy the bacterium. Our research has shown that pathogenic Escherichia coli O86a:K61 secretes the protease OmpT as part of outer membrane vesicles (OMVs), which inactivates coagulation Factor V and in so doing, induces a hemorrhagic state. To further characterize the effect of OmpT on the coagulation, this study has employed a genetic approach using wild type, an OmpT deletion mutant, and an over-expressing OmpT construct in pathogenic E. coli O157:H7. Although the growth of the three strains in liquid culture was not significantly different over time in nutrient rich media, OmpT over expression retarded cell growth in nutrient deficient media (p<0.05). Wild type cells and OmpT over expressing cells produced significantly larger numbers of different size ranges of outer membrane vesicles than OmpT deletion mutant (p<0.05). Wild type cells and outer membrane vesicles prolonged both the prothrombin time and activated partial thromboplastin time in normal human plasma, and this effect was enhanced with OmpT over expressing cells and abolished in the OmpT deletion mutant. Wild type cells and outer membrane vesicles inactivated Factor V, but not other factors (fibrinogen, FII and FX), in normal human plasma to a 250kDa product and the effect was increased in OmpT overexpressing strain and abolished in the OmpT deletion mutant. The effect of OmpT was direct and did not involve the host plasminogen system. In summary, this research indicates that OmpT has a role in OMV biogenesis and composition, and disrupting the coagulation process and fibrin barrier formation during the host innate immune response and in doing so may permit enhanced bacterial survival within host environments. The research will also lead to a greater understanding of the mechanism of action of E. coli virulence factors and positively impact healthcare environments through the development of novel and robust vaccines, antimicrobial drugs, diagnostic methods, and medical treatments.