Role for Host Exocytosis in Entry of the Bacterial Pathogen Listeria monocytogenes into Human Cells
Listeria monocytogenes (Lm) is a Gram-positive bacterium responsible for gastroenteritis, meningitis, and abortion in pregnant women. This intracellular pathogen is able to induce its internalization into host mammalian cells by expressing the bacterial surface protein, Internalin B (InlB). InlB binds to the Met receptor tyrosine kinase on the surface of host cells, resulting in activation of this receptor. Met activation leads to rearrangements of the actin cytoskeleton and host plasma membrane, resulting in uptake of Lm. Apart from actin polymerization, roles for other host physiological processes in Lm entry are unknown. Here, I demonstrate that host exocytosis, the fusion of host internal membrane vesicles with the plasma membrane, plays a critical role in InlB-mediated entry of Lm. Exocytosis is mediated by SNARE proteins, which promote the fusion of vesicles with the plasma membrane. Importantly, depletion of the human SNARE proteins VAMP3, STX4, or SNAP23 through RNA interference (RNAi) impaired entry of Listeria. In addition, experiments involving a probe consisting of VAMP3 fused to Green Fluorescent Protein (GFP) indicated that InlB-dependent entry was accompanied by focal exocytosis. Experiments involving RNAi-mediated depletion of Met showed that exocytosis requires this host Met receptor. RNAi-mediated depletion of host SNARE proteins failed to impair actin accumulation during internalization, suggesting that exocytosis and actin cytoskeletal rearrangement are separable host responses. Interestingly, SNARE proteins were needed for delivery of human GTPase Dynamin 2 to sites of InlB-mediated internalization. In addition, Dynamin 2 was required for entry of Lm. Collectively, my data identify exocytosis as a novel host physiological process exploited by Lm to promote infection.
Advisor: Ireton, Keith
Degree Name: Doctor of Philosophy
Degree Discipline: Department of Microbiology and Immunology
Publisher: University of Otago
Keywords: exocytosis; Listeria monocytogenes; InlB-medated entry; SNARE proteins; exocyst complex
Research Type: Thesis