INRAE, Université de Tours, ISP, F-37380, Nouzilly, France.
Present Address: Service Biologie Vétérinaire et Santé Animale, Inovalys, Angers, France.
BMC Microbiol. 2021 May 21;21(1):153. doi: 10.1186/s12866-021-02187-1.
Salmonella can invade host cells via a type three secretion system called T3SS-1 and its outer membrane proteins, PagN and Rck. However, the mechanism of PagN-dependent invasion pathway used by Salmonella enterica, subspecies enterica serovar Typhimurium remains unclear.
Here, we report that PagN is well conserved and widely distributed among the different species and subspecies of Salmonella. We showed that PagN of S. Typhimurium was sufficient and necessary to enable non-invasive E. coli over-expressing PagN and PagN-coated beads to bind to and invade different non-phagocytic cells. According to the literature, PagN is likely to interact with heparan sulfate proteoglycan (HSPG) as PagN-mediated invasion could be inhibited by heparin treatment in a dose-dependent manner. This report shows that this interaction is not sufficient to allow the internalization mechanism. Investigation of the role of β1 integrin as co-receptor showed that mouse embryo fibroblasts genetically deficient in β1 integrin were less permissive to PagN-mediated internalization. Moreover, PagN-mediated internalization was fully inhibited in glycosylation-deficient pgsA-745 cells treated with anti-β1 integrin antibody, supporting the hypothesis that β1 integrin and HSPG cooperate to induce the PagN-mediated internalization mechanism. In addition, use of specific inhibitors and expression of dominant-negative derivatives demonstrated that tyrosine phosphorylation and class I phosphatidylinositol 3-kinase were crucial to trigger PagN-dependent internalization, as for the Rck internalization mechanism. Finally, scanning electron microscopy with infected cells showed microvillus-like extensions characteristic of Zipper-like structure, engulfing PagN-coated beads and E. coli expressing PagN, as observed during Rck-mediated internalization.
Our results supply new comprehensions into T3SS-1-independent invasion mechanisms of S. Typhimurium and highly indicate that PagN induces a phosphatidylinositol 3-kinase signaling pathway, leading to a Zipper-like entry mechanism as the Salmonella outer membrane protein Rck.
沙门氏菌可以通过一种称为 T3SS-1 的 III 型分泌系统及其外膜蛋白 PagN 和 Rck 入侵宿主细胞。然而，沙门氏菌亚种肠炎沙门氏菌中依赖 PagN 的入侵途径的机制仍不清楚。
在这里，我们报告 PagN 在不同种属和亚种的沙门氏菌中高度保守且广泛分布。我们表明，肠炎沙门氏菌的 PagN 足以使非侵袭性的过表达 PagN 的大肠杆菌和涂有 PagN 的珠粒结合并入侵不同的非吞噬细胞。根据文献，PagN 可能与肝素硫酸蛋白聚糖（HSPG）相互作用，因为肝素处理可以以剂量依赖的方式抑制 PagN 介导的入侵。本报告表明，这种相互作用不足以允许内化机制。β1 整合素作为共受体的作用研究表明，β1 整合素基因缺陷型的鼠胚胎成纤维细胞对 PagN 介导的内化作用的允许性降低。此外，用抗β1 整合素抗体处理糖基化缺陷型 pgsA-745 细胞可完全抑制 PagN 介导的内化，支持β1 整合素和 HSPG 共同诱导 PagN 介导的内化机制的假说。此外，使用特异性抑制剂和表达显性失活衍生物表明，酪氨酸磷酸化和 I 类磷酸肌醇 3-激酶对触发 PagN 依赖性内化至关重要，就像 Rck 内化机制一样。最后，用感染细胞进行扫描电子显微镜检查显示，微绒毛样延伸具有拉链样结构的特征，吞噬涂有 PagN 的珠粒和表达 PagN 的大肠杆菌，如在 Rck 介导的内化过程中观察到的那样。
我们的结果提供了对肠炎沙门氏菌 T3SS-1 非依赖性入侵机制的新认识，并强烈表明 PagN 诱导了一种磷酸肌醇 3-激酶信号通路，导致了类似于沙门氏菌外膜蛋白 Rck 的拉链样进入机制。
