In situ analysis of type III secretion chaperone proteins indicates a cytosolic handover of virulence effectors.

Gram-negative bacteria can use type III secretion systems to inject effector proteins into eukaryotic target cells. Most effectors are co-expressed with specific chaperone proteins that are required for the secretion of their cognate effector. Although chaperones share characteristics across species, no common mechanism of action has been identified. In particular, it remains unclear, if and how chaperones target effectors to the type III secretion injectisome. In this study, we analyzed the interaction network, cellular localization, mobility, and function of SycH and SycE, two Yersinia enterocolitica T3SS chaperones, in live bacteria. While both chaperones strongly interacted with their cognate effectors, SycH additionally bound two negative regulators, YscM1/2, whereas SycE consistently showed weak interactions and proximity to various other effectors. In contrast, the chaperones did not specifically interact with the injectisome or the cytosolic T3SS components that were recently found to shuttle effectors to the injectisome. Mobility measurements and single particle tracking support these findings. Taken together, our results indicate a handover of the effector YopH from its chaperone SycH to the shuttle complexes in the bacterial cytosol and raise the possibility that a similar mechanism applies to other effector/chaperone pairs .