On the role of the sorting platform in hierarchical type III secretion regulation in enteropathogenic Escherichia...

The virulence of enteropathogenic Escherichia coli (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 Escherichia coli 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.