Understanding Legionella pneumophila's Interactions with Endocytic Rabs

The Legionella pneumophila bacterium is a respiratory pathogen phagocytized by alveolar macrophages. Through the use of a Dot/Icm type IV secretion system, Legionella is able to inject effector proteins into a host cell to ensure its survival. Secreted effector proteins allow the bacterium to evade degradation within the endocytic pathway as well as enable intracellular replication. Inhibiting lysosomal fusion for removal from the cell suggests an interaction between endocytic cell proteins and Legionella effector proteins. Previous studies have identified interactions between Dot/Icm substrates, bound to the membrane of a Legionella-containing vacuole, and Rab proteins localized to the late endosome and lysosome. In our lab, yeast two-hybrid assays have been used to examine interactions between early endosomal Rab proteins, Rab4a, Rab5a, Rab15, Rab22a, and Rab35, and various Legionella effector proteins. LegC7, known to disrupt vesicular trafficking and the endolysosomal pathway, was one of many Legionella effector proteins found to bind to Rabs in the yeast two-hybrid assay. I am examining the interaction between Legionella LegC7 and Rab5 proteins. Methods used to characterize these interactions include: amplifying genes of interest using PCR, GC Cloning, gel extractions, ligations, bacterial transformation, protein expression and DNA sequencing. Analyses of protein-protein binding will be investigated by GST- and Nickel-pull-down assays. Confirming the interactions between Legionella proteins and Rab GTPases will allow for a better understanding of how Legionella is able to modify the endocytic pathway using a Dot/Icm secretion system. This may provide insight into how bacteria manipulate host cells for survival.