Thesis

Confirmation and characterization of a potential Legionella pneumophila ARF-GAP

Legionella pnuemophila is an intracellular pathogen that is phagocytized by macrophage of our immune system. The bacterium uses a Type IV secretion system to secrete proteins across the phagosome membrane into the cytosol. Secreted effector proteins bind to host cell proteins to change the fate of the Legionella-containing phagosome (LCP) in two important ways. Legionella inhibits the endocytic pathway from fusing the LCP to endosomes/lysosomes, allowing the Legionella to escape proteolytic degradation. Furthermore, Rab1 and Arf1, exocytic host GTPases, are modulated by Legionella secreted proteins resulting in ER-derived vesicle fusion with the LCP creating a replicative niche for the Legionella. ARF1, a small GTPase that governs Golgi to ER retrograde transport, is switched "on" by a translocated Legionella effector protein, RalF, which acts as a guanine nucleotide exchange factor (GEF). A yeast two-hybrid screen in our laboratory identified several potential Arf1 GTPase activating proteins (GAP), potentially capable of switching ARF1 "off." Here, a putative Arf1 GAP, SamA (Sensory-box and ARFGAP Motifs), was shown to bind to ARF1 in a GST pull-down. An endpoint phosphate assay was developed and may be useful in determining GAP activity of SamA for ARF1. These data reveal a novel secreted effector protein that may aid in intracellular survival by modulating host cell trafficking.

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