Novel insights into the mechanism of SepL‐mediated control of effector secretion in enteropathogenic Escherichia coli

Type three secretion systems (T3SSs) are virulence determinants employed by several pathogenic bacteria as molecular syringes to inject effector proteins into host cells. Diarrhea‐producing enteropathogenic Escherichia coli (EPEC) uses a T3SS to colonize the intestinal tract. T3S is a highly coordinated process that ensures hierarchical delivery of three classes of substrates: early (inner rod and needle subunits), middle (translocators), and late (effectors). Translocation of effectors is triggered upon host‐cell contact in response to different environmental cues, such as calcium levels. The T3S substrate specificity switch from middle to late substrates in EPEC is regulated by the SepL and SepD proteins, which interact with each other and form a trimeric complex with the chaperone CesL. In this study, we investigated the link between calcium concentration and secretion regulation by the gatekeeper SepL. We found that calcium depletion promotes late substrate secretion in a translocon‐independent manner. Furthermore, the stability, formation, and subcellular localization of the SepL/SepD/CesL regulatory complex were not affected by the absence of calcium. In addition, we demonstrate that SepL interacts in a calcium‐independent manner with the major export gate component EscV, which in turn interacts with both middle and late secretion substrates, providing a docking site for T3S. These results suggest that EscV serves as a binding platform for both the SepL regulatory protein and secreted substrates during the ordered assembly of the T3SS. In this manuscript, we investigated the link between calcium concentration and regulation of effector secretion by the gatekeeper protein SepL in the type III secretion system of enteropathogenic Escherichia coli. Our results show that calcium depletion promotes effector secretion, though in a translocon‐independent manner. In addition, we demonstrate that the SepL/SepD/CesL complex does not participate in the calcium‐signaling pathway that leads to effector secretion in the absence of calcium. Moreover, we found that SepL interacts with the major export gate component EscV, and that this protein provides a docking site for type III secretion substrates.