The path and regulation of the type III secretion effector export

The type III secretion system (T3SS) is an essential virulence factor of many pathogenic bacteria including Yersinia enterocolitica. This nanomachine injects effector proteins and delivers them into the cytoplasm of the target eukaryotic cells, where they modulate the immune responses of the host in order to promote bacterial colonization and infection. Although the central feature of T3SS is effector export, the critical steps of the secretion process such as the selection and recruitment of effector proteins are still poorly understood. Previous studies suggest that the sorting platform plays a role in recruiting effectors to the machinery and helps in establishing export hierarchy. These conserved proteins organize into dynamic shuttle pod (sorting platform) structures at the cytosolic interface of the injectisome. To address the link between effector export and the dynamic nature of the cytosolic sorting platform, we performed a series of in vivo biochemistry techniques. Based on our single particle tracking and proximity labelling data, we found that the dynamic cytosolic T3SS components SctQ and SctL specifically bind to effectors in live bacteria during secretion. This complex then shuttles and targets the effectors to the membrane-bound injectisome, likely directing them to the export gate for subsequent secretion. Moreover, some T3SS effectors are often co-expressed with their cognate chaperone proteins and are required for effector export; however, no common mechanism of action has been found. Hence, we explored whether chaperone proteins contribute to the targeting of effectors into the injectisome. Since mapping the interactions of chaperone proteins will shed light on their function in effector export, we performed protein-protein interaction studies. Our data revealed strong interaction of chaperones to their respective effector proteins and additional T3SS components including the small negative regulators YscM1/2. Most importantly, our results notably failed to detect enrichment of the cytosolic sorting platform components (SctQ/K/L/N), implying that there is no direct interaction with chaperone proteins in live Yersinia. Further evidence came from the observation that SycH's mobility remained unchanged in the absence of the sorting platform complex based on our single particle tracking data. Thus, chaperones do not form a complex with the sorting platform components. Taken together, our findings revealed that chaperone dimer binds to a single effector in the cytosol, keeping them in a secretion-competent state, and transfers them to specific components of the shuttle pods (SctQ and SctL), which in turn delivers the cargo to the membrane-bound T3SS injectisome.