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Abstract：
The gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5\' kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process.IMPORTANCEStaphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5β1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.
摘要：
革兰氏阳性菌金黄色葡萄球菌可以通过与血浆蛋白纤连蛋白结合以利用宿主细胞整合素来侵入非专业吞噬细胞。通过磷脂酰肌醇-5'激酶的整联蛋白相关同种型,磷脂酰肌醇-4,5-二磷酸(PI-4,5-P2)的局部产生促进了这些病原体的整联蛋白介导的内化。在这项研究中,我们讨论了PI-4,5-P2定向磷酸酶对金黄色葡萄球菌内化的作用.ShRNA介导的单个磷酸肌醇5-磷酸酶的敲低显示突触蛋白1(SYNJ1)抵抗金黄色葡萄球菌对哺乳动物细胞的侵袭。的确,shRNA介导的耗竭以及通过CRISPR/Cas9对突触素1的遗传缺失导致关于整合素介导的摄取的功能获得表型。令人惊讶的是,Synj1-KO细胞中整合素的表面水平略有下调。然而，这些细胞显示PI-4,5-P2的局部积累增强,金黄色葡萄球菌的内化增加.虽然整合素相关蛋白酪氨酸激酶FAK的磷酸化水平没有改变，整合素结合和激活蛋白talin在突触素1敲除细胞中的金黄色葡萄球菌附近富集.扫描电子显微镜显示，在没有突触素1的情况下，膜内陷扩大，这解释了这些细胞内化整合素结合的微生物的能力增强。重要的是,Synj1-KO细胞的增强摄取和夸大的形态特征是通过野生型酶而不是磷酸酶失活突变体的重新表达来挽救的。因此，突触素1活性限制了整合素介导的金黄色葡萄球菌的侵袭，证实了PI-4,5-P2在此过程中的重要作用。重要金黄色葡萄球菌，一种重要的细菌病原体，可以通过捕获宿主纤连蛋白和参与整联蛋白α5β1侵入非专业吞噬细胞。了解金黄色葡萄球菌如何利用这种细胞粘附受体进行有效的细胞进入也可以阐明通过内吞作用对整合素的生理调节。以前的研究发现，一种特定的膜脂，磷脂酰肌醇-4,5-双磷酸酯(PIP2),支持内化过程。这里,我们扩展了这些发现，并报告了PIP2的局部水平受PIP2导向的脂质磷酸酶Synaptojanin1的活性控制。通过在细菌-宿主细胞附着位点去磷酸化PIP2，Synaptojanin1抵消整合素介导的微生物摄取。因此,我们的研究不仅为病原细菌对细胞受体的颠覆提供了新的见解，而且还强调了宿主细胞蛋白作为细菌侵入质膜的限制因子的作用。
