Semester

Fall

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Joan C Olson

Abstract

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa (Pa) is responsible for causing disease of high morbidity and mortality in individuals who are immunocompromised, suffer from damaged epithelial barriers, and who have cystic fibrosis. However, the underlying host cell compromise that allows the establishment of Pa infections remains unknown and is of importance to the development of novel strategies that interrupt Pa infections. The type III secretion system (T3S) allows the direct translocation of bacterial effectors into the host cell cytosol, and in this study was used first, to identify host cell properties involved in the initiation of Pa infections, and then second, to dissect the Pa infectious process. Using the T3S effector ExoS to monitor T3S translocation and the initiation of Pa infections in HT-29 epithelial cells, we identified a relationship between leading edge focal complex adhesion properties involved in cell migration and sensitivity to Pa-T3S. Highly migratory T24 epithelial cells were used to further explore the role of host cell migration in Pa infections, using manipulations of ExoS GTPase activating (GAP) and ADP-ribosyltransferase (ADPRT) activities to manipulate T3S. These studies allowed dissection of the Pa infectious process, and found that Pa expressing wild type ExoS preferentially bound to the leading edge of T24 cells, where ExoS GAP activity interfered with Pa internalization, and ExoS ADPRT activity interrupted actin-plasma membrane associations required for T3S translocation. Interestingly, both toxic events limited the Pa infectious process. Further studies of MTC, MTLn3, and MDCK epithelial cells identified a reciprocal relationship between Rac1 and Rho activation at the leading edge and Pa internalization and T3S translocation efficiency. Together, the studies in this dissertation highlight the role of actin-plasma membrane associations and Rho-GTPases in directing T3S translocation and the Pa infectious process, and the ability of Pa to hijack these factors during cell migration. Our studies in turn are consistent with cell migration properties induced in response to tissue damage being the cellular compromise that leads to the initiation of Pa infections, and that effectors such as ExoS are able to interrupt these same properties to limit the infectious process and maintain the opportunistic nature of Pa infections.

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