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. IMPORTANCE: Enteropathogenic 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.

The type III secretion system (T3SS) is a complex molecular device used by several pathogenic bacteria to translocate effector proteins directly into eukaryotic host cells. One remarkable feature of the T3SS is its ability to secrete different categories of proteins in a hierarchical manner, to ensure proper assembly and timely delivery of effectors into target cells. In enteropathogenic Escherichia coli, the substrate specificity switch from translocator to effector secretion is regulated by a gatekeeper complex composed of SepL, SepD, and CesL proteins. Here, we report a characterization of the CesL protein using biochemical and genetic approaches. We investigated discrepancies in the phenotype among different cesL deletion mutants and showed that CesL is indeed essential for translocator secretion and to prevent premature effector secretion. We also demonstrated that CesL engages in pairwise interactions with both SepL and SepD. Furthermore, while association of SepL to the membrane does not depended on CesL, the absence of any of the proteins forming the heterotrimeric complex compromised the intracellular stability of each component. In addition, we found that CesL interacts with the cytoplasmic domains of the export gate components EscU and EscV. We propose a mechanism for substrate secretion regulation governed by the SepL/SepD/CesL complex.

Salmonella enterica utilizes a virulence-associated type III secretion system (T3SS) to inject bacterial effectors directly into host cells. Central to this machinery is the sorting platform (SP), a cytosolic assembly whose core scaffolding protein, SpaO, is produced in two isoforms: a full-length (SpaO ^L ) and a shorter variant (SpaO ^short ) comprising the C-terminal 101 residues of SpaO ^L . Although SpaO ^short is evolutionarily conserved across type III secretion systems, its precise function has remained elusive. Here, we combined a sensitive, real-time translocation assay with site-directed photo-crosslinking to inform the role of SpaO ^short in Salmonella SPI-1 T3SS. Quantitative translocation data show that while SpaO ^short is not absolutely required for effector translocation, its absence significantly dampens T3SS-mediated protein delivery. Biochemical and structural probing further defined the interfaces between SpaO ^L and SpaO ^short , uncovering a previously unrecognized interaction mode between the two isoforms. Photo-crosslinking revealed that a single SpaO ^L molecule accommodates a SpaO ^short dimer via an N-terminal “docking motif,” an interaction that occurs in vivo while SpaO ^L is associated with other SP components. These results support a model in which SpaO ^short is integrated into the SP pods alongside SpaO ^L , OrgA, and OrgB, likely contributing to pod stabilization. Collectively, these findings provide new insights into how Salmonella and related bacteria assemble and maintain these specialized protein-injection systems. IMPORTANCE: Salmonella enterica is an increasing global public health threat. As part of its virulence arsenal, Salmonella relies on a type III secretion system (T3SS) or injectisome, a molecular injection device that translocates effector proteins into host cells to promote invasion and inflammation. A central component of this machine is the SpaO protein, which is produced in two forms: a full-length form and a shorter variant. Here, by studying the functional and structural relationship between the two SpaO forms in their native cellular environment, we define how and when they assemble within the injectisome. Employing quantitative injection assays in cultured cells, we define the shorter SpaO variant as an accessory structural piece that boosts effector delivery. These findings refine our understanding of injectisome assembly and function and provide mechanistic insight to inform future efforts to target T3SS-dependent pathogens through antivirulence strategies.

SUMMARY: Type III secretion systems are bacterial nanomachines specialized in protein delivery into target eukaryotic cells. The structural and functional complexity of these machines demand highly coordinated mechanisms for their assembly and operation. The sorting platform is a critical component of type III secretion machines that ensures the timely engagement and secretion of proteins destined to travel this export pathway. However, the mechanisms that lead to the assembly of this multi-component structure have not been elucidated. Herein, employing structure modeling and an extensive in vivo crosslinking strategy, we provide a detailed inter-subunit-contact survey of the entire sorting platform complex. Using the identified crosslinks as signatures for pairwise inter-subunit interactions in combination with systematic genetic deletions, we mapped the assembly process of this unique bacterial structure. Insights generated by this study could serve as the bases for the development of anti-virulence strategies to combat several medically important bacterial pathogens.