Listeria monocytogenesInlP interacts with afadin and facilitates basement membrane crossing

Abstract

ABSTRACTDuring pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such asListeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified theL. monocytogenesprotein Internalin P (InlP) as a secreted virulence factor critical for placental infection (1). Here, we show that InlP, but not the highly similarL. monocytogenesinternalin Lmo2027, binds to human afadin (encoded byAF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin’s involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited byAF-6knock-out MDCK cells.L. monocytogenes ΔinlPmutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion ininlP(inlPDLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promoteL. monocytogenestranscytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.