Introduction. Shigella flexneri is an intracellular bacterial pathogen that utilizes a type III secretion apparatus to inject effector proteins into host cells. Hypothesis/Gap Statement. The T3SS effector IpaH4.5 is important for the virulence of Shigella . Aim. This study aimed to elucidate the molecular mechanism and host target of the IpaH4.5 as well as its roles in S. flexneri infection. Methodology. The GAP assay was used to identify substrate Rab GTPases of IpaH4.5. A coimmunoprecipitation assay was applied to identify the interaction of Rab GTPases with IpaH4.5. A confocal microscopy analysis was used to assess the effects of IpaH4.5 on mannose 6-phosphate receptor (MPR) trafficking. To identify the effects of IpaH4.5 GAP activity on the activity of lysosomal cathepsin B, the Magic Red-RR assay was used. Finally, the intracellular persistence assay was used to identify IpaH4.5 GAP activity in S. flexneri intracellular growth. Results. We found that the effector IpaH4.5 disrupts MPR trafficking and lysosomal function, thereby counteracting host lysosomal degradation. IpaH4.5 harbours TBC-like dual-finger motifs and exhibits potent RabGAP activities towards Rab31. IpaH4.5 disrupts the transport of the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the Golgi to the endosome by targeting Rab31, thereby attenuating lysosomal function. As a result, the intracellular persistence of S. flexneri requires IpaH4.5 TBC-like GAP activity to mediate bacterial escape from host lysosome-mediated elimination. Conclusion. We identified an unknown function of IpaH4.5 and its potential role in S. flexneri infection.

中文翻译：

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志贺氏菌通过 IpaH4.5 灭活 Rab31 逃避溶酶体降解

介绍。 Shigella flexneri是一种细胞内细菌病原体，它利用 III 型分泌装置将效应蛋白注入宿主细胞。假设/差距陈述。T3SS 效应子 IpaH4.5 对志贺氏菌的毒力很重要。目的。本研究旨在阐明IpaH4.5的分子机制和宿主目标，以及其在角色S.菌感染。方法。 GAP测定用于鉴定IpaH4.5的底物Rab GTP酶。应用共免疫沉淀试验来鉴定 Rab GTP 酶与 IpaH4.5 的相互作用。共聚焦显微镜分析用于评估 IpaH4.5 对甘露糖 6-磷酸受体 (MPR) 运输的影响。为了鉴定 IpaH4.5 GAP 活性对溶酶体组织蛋白酶 B 活性的影响，使用了 Magic Red-RR 测定。最后，细胞内的持久性测定法用于鉴定IpaH4.5 GAP活性弗氏志贺菌的细胞内生长。结果。 我们发现效应子 IpaH4.5 会破坏 MPR 运输和溶酶体功能，从而抵消宿主溶酶体降解。IpaH4.5 具有类似 TBC 的双指图案，并对 Rab31 表现出强大的 RabGAP 活性。IpaH4.5 通过靶向 Rab31 来破坏阳离子依赖性甘露糖 6-磷酸受体 (CD-MPR) 从高尔基体到内体的转运，从而减弱溶酶体功能。因此，福氏链球菌的细胞内持久性需要 IpaH4.5 TBC 样 GAP 活性来介导细菌从宿主溶酶体介导的消除中逃逸。结论。我们确定了 IpaH4.5 的未知功能及其在福氏链球菌感染中的潜在作用。