Conserved salt bridges facilitate assembly of the helical core export apparatus of bacterial type III secretion systems

Virulence-associated type III secretion systems (T3SS) are utilized by Gram negative bacterial pathogens for injection of effector proteins into eukaryotic host cells. The transmembrane export apparatus at the core of T3SS is composed of a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU.These components comprise a number of highly conserved charged residues within their hydrophobic domains.The structure of theclosed state of the core complexSctR5S4T1revealed that several of these residues form inter- and intramolecular salt bridges that would have to be broken for pore opening. Mutagenesis of individual residues was shown to compromise assembly or secretion of both, the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Here we performed an in-depth mutagenesis analysis of these residues in the T3SS ofSalmonellaTyphimurium, coupled toblue native PAGE,in vivophotocrosslinking and luciferase-based secretion assays. Our data show thatthese conserved salt bridges are not critical for assembly of the respective protein but rather facilitate the incorporation of the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctSE44and SctTR204and the cytoplasmic domain of SctU in gating the T3SS injectisome. Overall, our analysis provides an unprecedented insight into the delicate requirements for the assembly and function of the machinery at the core of T3SS. Here you can find enclosed the simulation trajectories for the full SctFIRSTU and the SctIRT_WT, as well as SctIRT_K219A mutant. All trajectories are were generated according to protocol in the paper and are provided as Desmond. Frames or further analyses can be provided upon request direct to T.K.