The virulence-associated type III secretion system (T3SS), also known as the injectisome, is utilized by many gram-negative bacteria to translocate various effector proteins into host cells. Comprising around 200 subunits, the mega-dalton sized injectisome undergoes a highly coordinated assembly initiated by the export apparatus (EA). The EA serves as a scaffold for the inner and outer membrane rings, forming the base of the injectisome. Subsequently, the cytoplasmic compartment and needle filament are assembled.

The EA is composed of five conserved proteins, SctRSTUV, in a 5:4:1:1:9 stoichiometry, forming a unique helical cup structure. Its assembly involves distinct steps: initial assembly of pentameric SctR with one SctT, followed by recruitment of four SctS wrapping around SctR5T1 to form the core EA, and final addition of SctU and nonameric SctV to complete the assembly.

Given the size and complexity of the T3SS, its assembly is a complicated and energy-intensive task. Hence, elaborate regulatory networks exist to ensure robust assembly and prevent the waste of cellular resources. In our study, we observed that the EA has a highly conserved gene order across many T3SS-expressing pathogens. Interestingly, the assembly order (SctRTSU) does not follow the conserved gene order (sctRSTU). Matching assembly and gene orders is often observed in protein complex assemblies, as it allows co-translational assembly and minimizes the chance for subunit diffusion. We hypothesized that the conserved genetic organization may harbor regulatory motifs for robust EA assembly. In our analysis of Salmonella Typhimurium, we discovered that sctS and sctT are translationally coupled. sctS mRNA has a stem-loop structure which harbors the ribosome binding site of sctT, inhibiting de novo translation of SctT and allowing expression of SctT exclusively through the re-initiation mechanism. The nature of the upstream gene in the translational coupling was not shown to be a critical factor in the regulation of sctT expression and EA assembly. Our study presents the translational coupling between sctS and SctT as a crucial quality control mechanism in EA assembly.