Soto, J. E.; Wang, T.; Galan, J. E.; Lara-Tejero, M.

Salmonella enterica utilizes a virulence-associated type III secretion system (T3SS) to inject bacterial effectors directly into host cells. Central to this machinery is the sorting platform (SP), a cytosolic assembly whose core scaffolding protein, SpaO, is produced in two isoforms: a full-length (SpaOL) and a shorter variant (SpaOshort) comprising the C-terminal 101 residues of SpaOL. Although SpaOshort is evolutionarily conserved across type III secretion systems, its precise function has remained elusive. Here, we combined a sensitive, real-time translocation assay with site-directed photo-crosslinking to elucidate the role of SpaOshort in Salmonella SPI-1 T3SS. We found that while SpaOshort is not absolutely required for effector secretion, its absence significantly dampens T3SS-mediated protein delivery. Further biochemical and structural probing revealed that SpaOshort is a structural component of the sorting platform, arranged as a homodimer associated to SpaOL via an N-terminal \"docking motif.\" This interaction occurs while SpaOL is associated with other SP components, supporting a model in which SpaOshort is integrated into the SP pods alongside SpaOL, OrgA, and OrgB. Collectively, these findings show that SpaOshort, while not strictly essential, functions as a critical structural component of the sorting platform, providing new insights into how Salmonella and related bacteria assemble and maintain these specialized protein-injection systems.