Marcos-Vilchis Arely, Espinosa Norma, Alvarez Adrián F, Puente José L, Soto J Eduardo, González-Pedrajo Bertha
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Mexico.
J Bacteriol. 2025 Mar 20;207(3):e0044624. doi: 10.1128/jb.00446-24. Epub 2025 Mar 3.
The virulence of enteropathogenic (EPEC) depends on a type III secretion system (T3SS), a membrane-spanning apparatus that injects effector proteins into the cytoplasm of target enterocytes. The T3SS, or injectisome, is a self-assembled nanomachine whose biogenesis and function rely on the ordered secretion of three distinct categories of proteins: early, middle, and late type III substrates. In EPEC, this hierarchical secretion is assisted by several cytosolic protein complexes at the base of the injectisome. Among these, the sorting platform is involved in the recognition and sequential loading of the different classes of T3-substrates. In addition, a heterotrimeric gatekeeper complex, also known as a molecular switch, operates in concert with components of the T3SS export apparatus to guarantee the delivery of middle substrates prior to late substrate secretion. In this study, we showed that the sorting platform is differentially required for the secretion of distinct categories of substrates. Moreover, we demonstrated a cooperative interplay and protein-protein interactions between the sorting platform and the gatekeeper complex for proper middle and late substrate docking and secretion. Overall, our results provide new insights into the intricate molecular mechanisms that regulate protein secretion hierarchy during T3SS assembly.IMPORTANCEEnteropathogenic employs a type III secretion system to deliver virulence proteins directly into host cells, disrupting multiple cellular processes to promote infection. This multiprotein system assembles in a precise stepwise manner, with specific proteins being recruited and secreted at distinct stages. The sorting platform and the gatekeeper complex play critical roles in regulating this process, but their cooperative mechanism has not been fully elucidated. Here, we reveal a novel functional interaction between these two components, which is critical for hierarchical substrate recognition and secretion. These findings advance our understanding of the molecular mechanisms underlying bacterial virulence and suggest new potential targets for antimicrobial strategies aimed at disrupting T3SS function.
肠致病性大肠杆菌（EPEC）的毒力取决于III型分泌系统（T3SS），这是一种跨膜装置，可将效应蛋白注入靶肠上皮细胞的细胞质中。T3SS，即注射体，是一种自组装纳米机器，其生物发生和功能依赖于三类不同蛋白质的有序分泌：早期、中期和晚期III型底物。在EPEC中，这种分级分泌在注射体基部由几种胞质蛋白复合物协助。其中，分选平台参与不同类别的T3底物的识别和顺序加载。此外，一种异源三聚体守门人复合物，也称为分子开关，与T3SS输出装置的组件协同运作，以确保在晚期底物分泌之前输送中期底物。在本研究中，我们表明分选平台对于不同类别的底物分泌有不同的需求。此外，我们证明了分选平台与守门人复合物之间存在协同相互作用和蛋白质-蛋白质相互作用，以实现中期和晚期底物的正确对接和分泌。总体而言，我们的结果为T3SS组装过程中调节蛋白质分泌层次的复杂分子机制提供了新的见解。
重要性
肠致病性大肠杆菌利用III型分泌系统将毒力蛋白直接递送到宿主细胞中，破坏多种细胞过程以促进感染。这个多蛋白系统以精确的逐步方式组装，特定蛋白质在不同阶段被招募和分泌。分选平台和守门人复合物在调节这一过程中起关键作用，但其协同机制尚未完全阐明。在这里，我们揭示了这两个组件之间的一种新型功能相互作用，这对于分级底物识别和分泌至关重要。这些发现推进了我们对细菌毒力潜在分子机制的理解，并为旨在破坏T3SS功能的抗菌策略提出了新的潜在靶点。
