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Abstract

Type III secretion (T3S) systems are needle-like molecular machines that allow the injection of bacterial proteins directly into host cell cytosol or membranes in order to promote bacterial infection. Among the injected proteins are those containing transmembrane domains (TMD). Strongly hydrophobic TMD-effectors are prone to be mistargeted to the bacterial inner membrane unless they bind to a T3S chaperone (T3SC). This binding was found as a critical step for discrimination between: the classical Sec-dependent co-translational targeting and membrane insertion; and T3S, which is widely believed to be a post-translational process. Salmonella ́s pathogenicity island 2 encodes SscB and SseF which form a chaperone-effector complex. SscB was previously shown to be essential for the T3S of the TMD-effector SseF by avoiding its erroneous insertion into bacterial membranes. SscB binds to the chaperone binding domain (CBD) and first TMD of SseF. However, the processes underlying the targeting to T3S of TMD-effectors still remain elusive. Here, we aim to understand the features of SscB and the mode of interaction with SseF that ultimately prevent mistargeting. Analysis of the stability of SscB and SseF in Salmonella revealed co-stabilization since both proteins were more stable when interacting. Also, adding a SseF peptide (part of the CBD) stabilized purified SscB as observed by Nano-DSF. Furthermore, SscB has tetratricopeptide repeats and a circular dichroism spectrum typical of α-helical proteins. These features resemble those of class II T3SC which bind translocators, also TMD-proteins. However, according to the current chaperone classification, SscB should be classified as class I, since it binds a bona-fide T3S effector. Another feature reminiscent of the class II T3SC is the interaction of SscB with TMD and its ability to form dimers in solution, as observed by SEC-MALS. Moreover, the interacting partners of class II T3SC bear a "P/VXLXXP" consensus amino acid sequence in the CBD. This sequence is also present on SseF. Overall, these observations suggest that SscB-SseF interaction may occur during translation due to the observed co-stabilization and a need for rapid protection of TMD to avoid mistargeting to the Sec-dependent pathway. Also, it indicates that T3SC may have evolved to accommodate the structural characteristics (i.e. TMD) of the respective interacting partners.