Benioff Children's Hospital, University of California, San Francisco, San Francisco, California, United States of America.
Program in Microbial Pathogenesis and Host Defense, University of California, San Francisco, San Francisco, California, United States of America.
PLoS Pathog. 2018 May 30;14(5):e1007094. doi: 10.1371/journal.ppat.1007094. eCollection 2018 May.
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.
在妊娠期间，胎盘可保护胎儿免受母体免疫反应和细菌及病毒病原体的影响。李斯特菌等已经进化出特定机制来突破这一障碍的细菌病原体，为了解胎盘如何发挥其保护功能提供了独特的机会。我们之前已鉴定出李斯特菌蛋白 Internalin P（InlP）是一种重要的分泌毒力因子，对胎盘感染至关重要。在这里，我们表明，InlP 但不是高度相似的李斯特菌内部蛋白 Lmo2027，与人类 afadin（由 AF-6 编码）结合，后者是一种与细胞-细胞连接相关的蛋白质。InlP 的晶体结构揭示了几个独特的特征，包括一个扩展的富含亮氨酸重复（LRR）结构域和一个独特的 Ca2+结合位点。尽管 afadin 参与细胞-细胞连接的形成，但表达 InlP 的 MDCK 上皮细胞表现出其对可变形底物施加的牵引力的幅度降低，类似于 AF-6 敲除 MDCK 细胞表现出的牵引力降低。李斯特菌 ΔinlP 突变体在形成从极化上皮单层的基底表面突出的肌动蛋白丰富的突起的能力上存在缺陷，这是穿过这种单层（转胞吞作用）的必要步骤。表达 InlP 内部无义缺失的细菌（inlP ΔLRR5）也观察到类似的表型，该缺失特异性破坏了其与 afadin 的相互作用。然而，宿主细胞中 afadin 的缺失不能挽救转胞吞作用缺陷。我们得出的结论是，分泌的 InlP 靶向细胞质 afadin，特异性促进李斯特菌穿过上皮单层的基底表面的转胞吞作用，这可能有助于胎盘感染期间穿过基底膜。
