A role for ActA in epithelial cell invasion by Listeria monocytogenes

We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes. An in frame Delta actA mutant was const ructed in a hyperinvasive strain of prfA* genotype, in which all genes of t he PrfA-dependent virulence regulon, including actA, are highly expressed i n vitro. Loss of ActA production in prfA* bacteria reduced entry into Caco- 2, HeLa, MDCK and Vero epithelial cells to basal levels. Reintroduction of actA into the Delta actA prfA* mutant fully restored invasiveness, demonstr ating that ActA is involved in epithelial cell invasion. ActA did not contr ibute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes. Exp ression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and He pa 1-6 cells, indicating that ActA directs an internalization pathway speci fic for epithelial cells. Scanning electron microscopy of infected Caco-2 h uman enterocytes suggested that this pathway involves microvilli. prfA* bac teria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro) or prfA* Delta actA bacteria, efficiently invaded differen tiated Caco-2 cells via their apical surface. Microvilli played an active r ole in the phagocytosis of the prfA* strain, and actA was required for thei r remodelling into pseudopods mediating bacterial uptake. Thus, ActA appear s to be a multifunctional virulence factor involved in two important aspect s of Listeria pathogenesis: actin-based motility and host cell tropism and invasion.