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 the closed state of the core complex SctR₅S₄T₁ revealed that several of these residues form inter- and intramolecular salt bridges, some of which 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 of Salmonella Typhimurium, coupled to blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these 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 SctS_E44 and SctT_R204 and 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.

毒力相关的 III 型分泌系统 (T3SS) 被革兰氏阴性细菌病原体用于将效应蛋白注射到真核宿主细胞中。T3SS 核心的跨膜输出装置由疏水蛋白 SctR、SctS、SctT 和 SctU 的独特螺旋复合物组成。这些成分在其疏水结构域内包含许多高度保守的带电残基。核心复合体封闭态的结构 SctR ₅ S ₄ T ₁揭示这些残基中的一些形成分子间和分子内盐桥，其中一些必须被破坏才能打开孔。单个残基的诱变会损害毒力相关和相关鞭毛 T3SS 的组装或分泌。然而，这些保守的带电残基在 T3SS 的组装和功能中的确切作用仍然难以捉摸。在这里，我们对鼠伤寒沙门氏菌T3SS 中的这些残基进行了深入的诱变分析，与蓝色天然 PAGE 结合，在体内光交联和基于荧光素酶的分泌测定。我们的数据表明，这些保守的盐桥对于相应蛋白质的组装并不重要，而是有助于将以下亚基整合到组装复合物中。我们的数据还表明，这些保守的带电残基对于 III 型依赖性分泌至关重要，并揭示了 SctS _E44和 SctT _R204与_SctU细胞质域之间在 T3SS 注射体门控中的功能联系。总体而言，我们的分析为 T3SS 核心机械的组装和功能的精细要求提供了前所未有的洞察力。