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Introduction: Type III secretion (T3S) systems are needle-like molecular machines that allow the transport of proteins across gram- negative bacteria membranes directly into the host cell to ultimately promote bacterial survival. Among the delivered proteins are those containing transmembrane domains (TMD). The presence of more hydrophobic TMD in T3S substrates requires the binding of a cognate T3S chaperone (T3SC) that allows correct targeting to T3S and avoids incorrect targeting to bacterial membranes by outcompeting from recognition by the components of the Sec-pathway. Thus, a precise orchestration of these processes is crucial for the efficient interaction of the chaperone/TMD-substrate pairs. Here, we aim to unveil the key features and processes that underlie T3S of TMD-substrates using as a model the Salmonella"s T3S chaperone/TMD-effector, SscB/SseF. Methods: For the biophysical characterization of SscB and SscB/SseF, the purified proteins were analyzed by CD, Nano-DSF and SEC-MALS. To determine the key residues for complex formation was used alanine scanning mutagenesis. Furthermore, protein interactions were analyzed by in vivo photocrosslinking and protein stability in Salmonella using a chloramphenicol-based assay. To assess T3S, a Nanoluc luciferase-based assay was developed for SPI-2 (Salmonella"s pathogenicity island 2) inducing conditions. Results: Previously, it was observed that Salmonella"s TMD-effector SseF required its cognate T3SC SscB to avoid erroneous insertion into bacterial membranes and to allow correct targeting to T3S. These proteins are encoded adjacently in SPI-2 (sscB-sseF), and when this order is changed a decrease in the T3S of SseF was observed, both in SPI-2 inducing conditions and during infection. Moreover, SscB was stabilized by SseF in Salmonella, and the presence of the chaperone binding domain (CBD) of SseF was sufficient for stabilizing purified SscB. The same was observed in SseF which requires SccB to be stabilized. Furthermore, SscB interacts with SseF"s CBD domain and first TMD, but not with the second TMD, in a stoichiometry of 1:1. Interestingly, SscB structural features resemble those of T3SC that bind translocators, also TMD-proteins. Furthermore, by performing alanine scanning mutagenesis, it was shown that SseF bore a "P/VXLXXP" consensus amino acid sequence in the CBD, which is conserved in the translocators of Salmonella and other bacterial species. Additionally, other Salmonella"s T3SC of translocators, although not able to promote SseF secretion, could stabilize SseF. Discussion: Overall, these observations suggest that formation of SscB/SseF may occur rapidly since a specific gene organization is required and the proteins co-stabilized. This may be to allow for rapid protection of TMD and thus avoid mistargeting to the Sec-dependent pathway. Additionally, these results have shown that T3SC may have evolved to accommodate the structural characteristics of their respective interacting partners.
