Role of the host cell in the type III translocation of Pseudomonas aeruginosa exoenzyme S

Exoenzyme S (ExoS) is a bifunctional toxin that is directly translocated into eukaryotic cells by the Pseudomonas aeruginosa (Pa) type III secretory (T3S) process. The N-terminal region of ExoS includes a GTPase activating protein (GAP) activity and the C-terminal region includes an ADP-ribosyltransferase (ADPRT) activity. The T3S-translocation (T3S-T) of ExoS allows the bacteria to directly manipulate host cell function, thereby contributing the virulence of Pa. A cell culture model of infection was developed to study the effects of ExoS on cell function, which differentiates the effects of ExoS expression from other bacterial effects. Previous studies found that ExoS inactivates cell function, and this inactivation was largely attributed to the ADPRT activity. Bacterially translocated ExoS (T3S-T-ExoS) was also found to ADP-ribosylate a number of low molecular weight G-proteins (LMWG-proteins) within the cell. While the effects of T3S-T-ExoS on cell function were characterized, the role of the host cell in the T3S process remained unknown. Studies described in this dissertation characterize host cell influences in the targeting of ExoS ADPRT activity to specific eukaryotic substrates, characterize host cell mechanism of resistance to ExoS, and examine mechanisms associated with differences in host cell influence on T3S-T-ExoS ADPRT activity. Results from these studies identified two main patterns of T3S-T ExoS ADP-ribosylation of eukaryotic proteins, a model system to examine the host cell influence on the Pa T3S-T process, and a method of tracking intracellular ExoS by examining ExoS-eukaryotic protein interactions. Combined, these studies expand previous knowledge about the host cell influence on the Pa T3S-T process and provide methods to understand host cell mechanisms targeting T3S effector activity