The modulation of polymorphonuclear neutrophil function by cytotoxic necrotizing factor type 1 -- expressing uropathogenic Escherichia coli /

by Davis, Jon Michael.

Title of Dissertation: The Modulation of Polymorphonuclear Neutrophil Function by Cytotoxic Necrotizing Factor Type 1 - Expressing Uropathogenic Escherichia coli Jon M. Davis, Doctor of Philosophy, 2005 Thesis Directed by: Alison D. O’Brien, Ph.D. Professor, Department of Microbiology and Immunology Uropathogenic Escherichia coli (UPEC) cause more than 85% of all urinary tract infections (UTI). These infections primarily affect women, and over half of all women will experience at least one UTI in their lifetime. Animal models of UTI pathogenesis have provided some insight into the role of various UPEC virulence factors. In these animal studies, the toxin Cytotoxic Necrotizing Factor type 1 (CNF1) has been shown to have a significant role in the pathogenesis of UTI. One of the most striking features of CNF1-expressing UPEC infection in the in vivo models was magnitude of the acute iii inflammatory response. Compared to a cnf1 isogenic mutant, CNF1-expressing UPEC elicited an acute inflammatory response characterized by an extensive infiltration of polymorphonuclear leukocytes (PMN) into tissue infected with CNF1-expressing UPEC. In spite of the enhanced acute inflammatory response, the CNF1-expressing UPEC had a significant survival advantage compared to the cnf1 isogenic mutant. CNF1 is an AB toxin that deamidates the catalytically-active glutamine residue in the Rho family of small GTPases. The Rho family GTPases are intracellular signaling molecules responsible for the control of many cellular function in eukaryotic cells such as PMNs. In PMNs, Rho GTPases control the processes of phagocytosis and chemotaxis in addition to the generation of reactive oxygen species (ROS). These observations formed the foundation of the hypothesis that CNF1-expressing UPEC modulate PMN function. This hypothesis was tested in vitro with elicited mouse PMNs and showed that CNF1expressing UPEC modulate the antimicrobial response of PMNs through several mechanisms. First, CNF1-expressing UPEC alter the capacity of PMNs to remodel their plasma membrane and cluster CD11b in response to serum-opsonized UPEC which leads to a diminished PMN phagocytic capacity. Second, CNF1 expressed by UPEC results in an increase in the amount of Rac2 in PMNs. Third, PMNs co-incubated with CNF1expressing UPEC generate an enhanced intracellular ROS. Finally, CNF1-expressing UPEC release CNF1 via outer membrane vesicles that interact with the PMN membrane. These results support the hypothesis that CNF1 is a UPEC virulence factor and lead to a new molecular model of UPEC interaction with the host innate immune system. iv The Modulation of Polymorphonuclear Neutrophil Function by Cytotoxic Necrotizing Factor Type 1 – Expressing Uropathogenic Escherichia coli By Jon Michael Davis Dissertation submitted to the Faculty of the Department of Microbiology and Immunology Graduate Program of the Uniformed Services University of the Health Sciences F. Edward Hèbert School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2005 v