Staphylococcus aureus internalization by non-professional phagocytes is considered a main pathogenicity mechanism leading to chronic infections. The well-established mechanism of Staphylococcus aureus internalization is mediated by fibronectin (Fn)-binding proteins (FnBPs), Fn as a bridging molecule and the host cell α₅β₁ integrin. We previously identified a novel alternative internalization mechanism in Staphylococcus aureus, which involves the major autolysin Atl and the host cell heat shock cognate protein 70 (Hsc70). Atl-dependent internalization is also employed by the coagulase-negative Staphylococcus epidermidis, where it might represent the major or even sole internalization mechanism, because of the lack of FnBP-homologous proteins. In this study, we aimed to further characterize the Atl-dependent staphylococcal internalization mechanism. We performed biomolecular interaction analysis (BIA) to quantify the adhesive properties of Atl and found multivalent and high affinity interactions of Atl with Fn and Hsc70. Confocal laser scanning microscopy (CLSM) and a flow-cytometric internalization assay in combination with different pharmacological inhibitors suggested an involvement of the α₅β₁ integrin, Fn and Hsc70 and subsequent signaling events mediated by Src and phosphoinositide 3 (PI3) kinases in the Atl-dependent staphylococcal uptake by EA.hy 926 cells. Further characterization of the endocytic machinery implicated a role for clathrin-dependent receptor-mediated endocytosis involving actin cytoskeletal rearrangements and microtubules. In conclusion, Atl ubiquitous among staphylococcal species may substitute for the FnBPs ensuring low-level internalization via a mechanism that seems to share important features with the FnBP-mediated staphylococcal uptake potentially being the prerequisite for the development of therapy-resistant chronic infections by staphylococcal strains that lack FnBPs.

非专业吞噬细胞对金黄色葡萄球菌的内在化被认为是导致慢性感染的主要致病机制。行之有效的机制金黄色葡萄球菌内由纤连蛋白（FN） -结合蛋白（FnBPs）介导的，FN作为桥联分子和α宿主细胞₅ β ₁整联蛋白。我们先前在金黄色葡萄球菌中发现了一种新的替代内在化机制，其中涉及主要的自溶素Atl和宿主细胞热休克同源蛋白70（Hsc70）。凝固酶阴性表皮葡萄球菌也采用Atl依赖的内化作用，由于缺乏FnBP同源蛋白，它可能代表主要或唯一的内部化机制。在这项研究中，我们旨在进一步表征Atl依赖的葡萄球菌内在化机制。我们进行了生物分子相互作用分析（BIA），以量化Atl的粘附性能，并发现Atl与Fn和Hsc70的多价和高亲和力相互作用。共聚焦激光扫描显微镜（CLSM），并与不同的药理学抑制剂组合的流式细胞内化测定建议的α的参与₅ β ₁整合素，Fn和Hsc70以及由EA.hy 926细胞摄取的Atl依赖性葡萄球菌中Src和磷酸肌醇3（PI3）激酶介导的后续信号转导事件。内吞机制的进一步表征牵涉网格蛋白依赖受体介导的内吞作用，涉及肌动蛋白细胞骨架重排和微管。总之，在葡萄球菌中普遍存在的Atl可以替代FnBP，从而通过与FnBP介导的葡萄球菌摄取具有重要特征的机制来确保低水平的内在化，这可能是葡萄球菌菌株产生耐治疗性慢性感染的前提缺乏FnBP。