Analysis of the assembly of the virulence-associated Salmonella type III secretion system

Abstract

Salmonella enterica serovar Typhimurium utilizes the type III secretion system (T3SS) to directly translocate bacterial effector proteins into the host cytosol, thereby establishing infection and promoting intracellular survival within host cells. Although the overall architecture and function of the T3SS have been extensively characterized, the precise order of assembly of its substructures, particularly the base components, remains incompletely understood. Conflicting models propose either an outside-in assembly pathway initiated at the outer membrane via SctC (InvG) formation, followed by recruitment of the remaining basal body components SctD (PrgH) and SctJ (PrgK), or an inside-out process originating at the inner membrane through assembly of the export apparatus, followed by recruitment of SctJ and SctD and finally SctC. The aim of this study was to clarify this conflict using a combination of in vivo photocrosslinking and luciferase-based secretion assays. Photocrosslinking experiments revealed that the interaction between SctD and SctJ remained detectable in the absence of SctC, although with reduced signal intensity compared to the wild type, indicating that SctD-SctJ assembly depends on prior assembly of SctC. Similarly, the SctD-SctC interaction was still detected in the absence of SctJ, albeit with reduced intensity, suggesting that stable SctD-SctC association depends on prior SctJ assembly. Interactions involving sorting platform components, including SctD-SctK and SctK-SctQ, also remained detectable in ΔsctC and ΔsctJ strains, indicating that early recruitment of cytoplasmic sorting platform components can occur before completion of the basal body. However, the reduced crosslinking signals observed in mutant backgrounds suggest that the fully assembled basal body contributes to stabilization of these interactions. Functional analyses using NanoLuc-based secretion assays further demonstrated that no increased accumulation of the early substrate InvJ occurred in the periplasm of ΔsctC strains, indicating that secretion initiation requires the presence of SctC. Together, our findings reconcile the previously proposed inside-out and outside-in assembly models by supporting a refined hybrid mechanism in which SctJ and SctC assemble independently at the inner and outer membranes, respectively, followed by insertion of SctD between both structures to complete basal body assembly.