Research Article: Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing

Date Published: May 30, 2018

Publisher: Public Library of Science

Author(s): Cristina Faralla, Effie E. Bastounis, Fabian E. Ortega, Samuel H. Light, Gabrielle Rizzuto, Lei Gao, Denise K. Marciano, Salvatore Nocadello, Wayne F. Anderson, Jennifer R. Robbins, Julie A. Theriot, Anna I. Bakardjiev, Renée M. Tsolis.

http://doi.org/10.1371/journal.ppat.1007094

Abstract

During 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 as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-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 by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants 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 in inlP (inlP ΔLRR5) 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 promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

Partial Text

During pregnancy, the consequences of placental infection can be severe, ranging from maternal sepsis to miscarriage, and can lead to pre-term birth and lifelong disability [1]. Fortunately, such infections are relatively rare–which stands as a testament to the strength of the feto-maternal barrier. Despite serving such an important function, the molecular, cellular and histological components of feto-maternal barrier have only just begun to be elucidated. Because the barrier is so effective at preventing infection, pathogens that do manage to cross it must have evolved strategies of countering host defenses and thus provide a unique opportunity for addressing the mechanistic features that make the feto-maternal barrier so formidable [2].

En route to infection of the fetus, L. monocytogenes experiences multiple bottlenecks, from trafficking to the placenta [2] to surviving the placental innate immune defenses [49] to crossing the trophoblast monolayer and its associated basement membrane into the fetal stroma and fetal circulation [19]. InlP initially attracted our interest due to its identification in a screen for L. monocytogenes mutants that were defective in their ability to infect the placenta [21]. Two unbiased screening methods to identify potential placental binding partners, a yeast two-hybrid screen using a placental cDNA library and mass spectrometric identification of human proteins pulled down by InlP from placental extracts, converged on afadin as a significant candidate binding partner. Afadin is not found on the cell surface, but instead is primarily associated with the cytoplasmic face of cell-cell junctions containing nectin [41, 50]. Therefore, it seems likely that InlP is most helpful in breaching the trophoblast monolayer and accessing the fetal stroma.

 

Source:

http://doi.org/10.1371/journal.ppat.1007094