The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Abstract

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

Functional dissection of translocon proteins of the Salmonella Pathogenicity Island 2-encoded type III secretion system

“…3). Deletions in sseB Δ5, sseB Δ6 affect the binding site for SseA that acts as chaperone for SseB and SseD [9]. The altered partitioning observed for strains expressing these alleles may be due to the altered binding of chaperone SseA to its targets and altered stability of these proteins.…”

“…Protein domains predicted as putative transmembrane regions or coiled-coil regions were deleted, as well as N- or C-terminal portions, and previously defined binding regions for the specific chaperone SseA [9,10]. …”

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

“…The characteristics of EscC are reminiscent of SseA of Salmonella SPI-2. SseA interacts with the C-terminal coiled-coil domain of SseB and is classified as a class III flagellar chaperone (Zurawski & Stein, 2004). Similarly, coiled-coil domains in the C-terminal regions of EseB and EseD were predicted with COILS.…”

Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2

“…6B), suggesting that the C-terminal coiled-coil domain of SsaE is important for secretion of the SPI-2 effectors. It has been reported that SseA functions as a chaperone for SseB and is required for stabilization of SseB in the bacterial cytosol and for SseB export to the bacterial surface (9,49,62,63). Therefore, to demonstrate the presence of the SseA-SseBSsaE protein complex in the Salmonella cytoplasm, the immunoprecipitation by FLAG pull-down assays using lysates from the Salmonella strain expressing SseA-2HA (chromosomal mutation) harboring either pSsaE-FLAG or pBAP-FLAG were carried out.…”

“…Efficient secretion of SseC and SseD is required for the presence of SseB, but SseB secretion is independent of these two translocators (31,42). Furthermore, stable expression of these translocators requires the chaperone SseA for SseB and SseD and a putative chaperone SscA for SseC (49,62,63).…”