Interplay between SpaO variants shapes the architecture of the Salmonella type III secretion sorting platform

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 (SpaO^L) and a shorter variant (SpaO^short) comprising the C-terminal 101 residues of SpaO^L. Although SpaO^short 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 SpaO^short in Salmonella SPI-1 T3SS. We found that while SpaO^short is not absolutely required for effector secretion, its absence significantly dampens T3SS-mediated protein delivery. Further biochemical and structural probing revealed that SpaO^short is a structural component of the sorting platform, arranged as a homodimer associated to SpaO^L via an N-terminal "docking motif." This interaction occurs while SpaO^L is associated with other SP components, supporting a model in which SpaO^short is integrated into the SP pods alongside SpaO^L, OrgA, and OrgB. Collectively, these findings show that SpaO^short, 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.