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 inform the role of SpaO^short in Salmonella SPI-1 T3SS. Quantitative translocation data show that while SpaO^short is not absolutely required for effector translocation, its absence significantly dampens T3SS-mediated protein delivery. Biochemical and structural probing further defined the interfaces between SpaO^L and SpaO^short, uncovering a previously unrecognized interaction mode between the two isoforms. Photo-crosslinking revealed that a single SpaO^L molecule accommodates a SpaO^short dimer via an N-terminal "docking motif," an interaction that occurs in vivo while SpaO^L is associated with other SP components. These results support a model in which SpaO^short is integrated into the SP pods alongside SpaO^L, OrgA, and OrgB, likely contributing to pod stabilization. Collectively, these findings provide new insights into how Salmonella and related bacteria assemble and maintain these specialized protein-injection systems.IMPORTANCESalmonella enterica is an increasing global public health threat. As part of its virulence arsenal, Salmonella relies on a type III secretion system (T3SS) or injectisome, a molecular injection device that translocates effector proteins into host cells to promote invasion and inflammation. A central component of this machine is the SpaO protein, which is produced in two forms: a full-length form and a shorter variant. Here, by studying the functional and structural relationship between the two SpaO forms in their native cellular environment, we define how and when they assemble within the injectisome. Employing quantitative injection assays in cultured cells, we define the shorter SpaO variant as an accessory structural piece that boosts effector delivery. These findings refine our understanding of injectisome assembly and function and provide mechanistic insight to inform future efforts to target T3SS-dependent pathogens through antivirulence strategies.