InlB Expedites Vacuole Escape and Intracellular Proliferation by Promoting Rab7 Recruitment via Vps34.

Rapid phagosomal escape mediated by listeriolysin O (LLO) is a prerequisite for Listeria monocytogenes intracellular replication and pathogenesis. Escape takes place within minutes after internalization from vacuoles that are negative to the early endosomal Rab5 GTPase and positive to the late endosomal Rab7. Using mutant analysis, we found that the listerial invasin InlB was required for optimal intracellular proliferation of L. monocytogenes. Starting from this observation, we determined in HeLa cells that InlB promotes early phagosomal escape and efficient Rab7 acquisition by the -containing vacuole (LCV). Recruitment of the class III phosphoinositide 3-kinase (PI3K) Vps34 to the LCV and accumulation of its lipid product, phosphatidylinositol 3-phosphate (PI3P), two key endosomal maturation mediators, were also dependent on InlB. Small interfering RNA (siRNA) knockdown experiments showed that Vps34 was required for Rab7 recruitment and early (LLO-mediated) escape and supported InlB-dependent intracellular proliferation. Together, our data indicate that InlB accelerates LCV conversion into an escape-favorable Rab7 late phagosome via subversion of class III PI3K/Vps34 signaling. Our findings uncover a new function for the InlB invasin in pathogenesis as an intracellular proliferation-promoting virulence factor. Avoidance of lysosomal killing by manipulation of the endosomal compartment is a virulence mechanism assumed to be largely restricted to intravacuolar intracellular pathogens. Our findings are important because they show that cytosolic pathogens like L. monocytogenes, which rapidly escape the phagosome after internalization, can also extensively subvert endocytic trafficking as part of their survival strategy. They also clarify that, instead of delaying phagosome maturation (to allow time for LLO-dependent disruption, as currently thought), via InlB L. monocytogenes appears to facilitate the rapid conversion of the phagocytic vacuole into an escape-conducive late phagosome. Our data highlight the multifunctionality of bacterial virulence factors. At the cell surface, the InlB invasin induces receptor-mediated phagocytosis via class I PI3K activation, whereas after internalization it exploits class III PI3K (Vsp34) to promote intracellular survival. Systematically elucidating the mechanisms by which interferes with PI3K signaling all along the endocytic pathway may lead to novel anti-infective therapies.