Institute for Infectiology, University of Münstergrid.5949.1, Münster, Germany.
Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Antimicrob Agents Chemother. 2021 Nov 17;65(12):e0095821. doi: 10.1128/AAC.00958-21. Epub 2021 Sep 20.
Infections with enteropathogenic Escherichia coli (EPEC) cause severe diarrhea in children. The noninvasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir (translocated intimin receptor). Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. In addition, these substances reduced bacterial binding to host cells, effector-dependent cell detachment, and abolished attaching and effacing lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and enterohemorrhagic E. coli infections.
肠致病性大肠杆菌（EPEC）感染可导致儿童严重腹泻。这种非侵袭性细菌黏附于小肠的肠细胞，并利用 III 型分泌系统（T3SS）将效应蛋白注入宿主细胞，从而改变和利用细胞过程，有利于细菌的存活和复制。多项研究表明，细菌病原体的 T3SS 对于毒力是必需的。此外，T3SS 介导的效应蛋白易位的丧失会导致细菌的免疫识别和清除增加。因此，T3SS 被认为是抗病毒策略和新型治疗药物开发的有前途的靶标。在这里，我们报告了基于 EPEC 效应蛋白 Tir（转位内膜受体）易位的高通量筛选测定的结果。使用该测定法，我们筛选了来自六个不同化合物文库的超过 13000 种小分子化合物，并鉴定出三种物质，它们对易位具有显著的剂量依赖性作用，而对细菌或真核细胞活力没有不良影响。此外，这些物质减少了细菌与宿主细胞的结合、效应蛋白依赖性细胞脱落，并消除了黏附和破坏损伤的形成，而不影响 T3SS 或相关效应蛋白的表达。此外，抑制剂对细菌运动或志贺毒素表达没有影响。总之，我们已经鉴定出三种可强烈抑制效应蛋白向哺乳动物细胞中 T3SS 介导易位的新型化合物，它们可能是治疗 EPEC 和肠出血性大肠杆菌感染的有价值的先导物质。
