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085 THE ROLE OF SMALL GTP-BINDING PROTEINS IN PHAGOSOME MATURATION NA Kaniuk1, WD Heo2, X Jiang1, V Canadien1, T Meyer2, JH Brumell1 Phagocytosis is a form of endocytosis that mediates the internalization of large particles into the cell, and plays an essential role in development and immunity. Phagosomes mature by interacting with the endosomal pathway and eventually fuse with lysosomes. Members of the Rab family of small GTPases are recruited to the phagosome and promote its maturation, however, it is not known what role other members of the GTPase superfamily (Ras, Rho and Arf/Sar family of proteins) have in phagosome maturation. My research aim is to identify and functionally characterize members of these families of GTPases that are important in phagosome maturation. To investigate what roles these proteins have, epithelial HeLa cells were infected with a non-invasive strain (deltainvA/Inv) of Salmonella enterica sv Typhimurium (S. Typhimurium). The S. Typhimurium deltainvA/Inv strain traffics to the lysosome, where it fails to replicate and is killed. HeLa cells transfected with 73 GFP/CFP-GTPases (representing Ras, Rho and Arf/Sar families) were infected with the deltainvA/Inv strain for various times to generate model phagosomes. Following uptake of the bacteria, GTPase association was tracked using immunofluorescence microscopy. A positive association of a GTPase resulted in a distinct ring around the intracellular bacteria. Of the 35 different Ras GTPases screened, preliminary observations indicate some novel Ras association (H-Ras, N-Ras, and RasL12) with the model phagosome. These finding are significant as a role in phagosome maturation in the Ras family has not been demonstrated.
1Cell Biology, The Hospital for Sick Children, Toronto, Ontario; 2Department of Molecular Pharmacology, Stanford Medical School, Stanford, California, USA
We also infected cells with wildtype S. Typhimurium and monitored association with the same 73 GTPases. Following invasion, the wildtype bacteria occupy a vacuole niche that does not fuse with lysosomes, allowing the bacteria to replicate. The mechanism by which these bacteria block lysosomal fusion is unknown, however, it has been speculated that they manipulate GTPase activity. By observing which GTPases interact with wildtype S. Typhimurium, we identified GTPases involved in trafficking of a pathogenic bacterium.
Localizing small GTPases to the model phagosome has provided new targets for studying the complex process of phagosomal maturation and identified GTPases that differ in recruitment to model phagosomes versus wildtype bacteria. This will provide insight into how S. Typhimurium modulates phagosome maturation to allow its intracellular growth and cause gastrointestinal disease.