Rab5a and RIN2 Promote L. monocytogenes Cell-to-Cell Spreading in JEG-3 Cells

Abstract

Abstract Background The facultative intracellular pathogen, Listeria monocytogenes, disseminates amongst host cells to infect entire organs. This cellular transfer is driven through the coordination of bacterial factors that form actin-rich finger-like listeriopod protrusions in “sending” cells and host cell endocytosis of these large structures in “receiving” cells. Previous studies have demonstrated that the process that internalizes these large structures exploits both Caveolin- and Flotillin-mediated endocytosis. However, how early endocytic regulators contribute to L. monocytogenes cell-to-cell spreading remains unexplored. Methods Rab5a/RIN2 localization was analyzed using immunofluorescence microscopy during L. monocytogenes infections in JEG-3 cells. The proteins functions was then examined using transfection with wild-type and mutant constructs and siRNA-mediated knockdown approaches. Results We show that Rab5a and RIN2 are selectively enriched at L. monocytogenes invaginations, but not at listeriopods. Functional cell-to-cell spreading assays coupled with loss- and gain-of-function approaches showed that depletion or inhibition of Rab5a in the receiving cells significantly reduced bacterial dissemination, whereas expression of constitutively active Rab5a enhanced cell-to-cell spreading. Similarly, RIN2 localized to Rab5a-positive invaginations and was required for efficient cell-to-cell spreading, with RIN2 knockdowns resulting in a significant reduction in bacterial transfer. Conclusion Our data identifies Rab5a and RIN2 as key host endocytic regulators that promote L. monocytogenes dissemination.