Assembly of a functional type III secretion system (T3SS) requires intricate protein-protein interactions in many bacterial species. In Vibrio parahaemolyticus, the leading cause of seafood-associated diarrheal illnesses, the gatekeeper protein VgpA is essential for T3SS2 to secrete its substrates. However, it is unknown if VgpA interacts with other core elements of T3SS2 to mediate its substrate secretion. Through bacterial two-hybrid (BACTH) analysis, we now show that VgpA physically interacts with VscN2 (an ATPase essential for T3SS function) and six other hypothetical proteins. Mutation of isoleucine to alanine at residue 175 of VgpA (VgpA^I175A) abolished its ability to interact with VscN2. Importantly, complementation of a VgpA nonsense mutant (vgpA’) with VgpA^I175A did not restore the ability of T3SS2 to secrete substrates, demonstrating that VgpA-VscN2 interaction is critical for the function of T3SS2. Bacterial cell fractionation and mass spectrometry analyses showed that vgpA’ resulted in significant alterations of T3SS2 protein abundance in multiple bacterial cell fractions. Particularly, VscN2 abundance in the inner membrane fraction and VscC2 abundance in the outer membrane fraction are significantly reduced in vgpA’ compared to those in WT. These results demonstrated that VgpA contributes to T3SS2 function via its interaction with VscN2 and possibly by affecting subcellular distribution of T3SS2 proteins.

功能性 III 型分泌系统 (T3SS) 的组装需要许多细菌物种中复杂的蛋白质-蛋白质相互作用。在副溶血弧菌（海鲜相关腹泻疾病的主要原因）中，看门蛋白 VgpA 对于 T3SS2 分泌其底物至关重要。然而，尚不清楚VgpA是否与T3SS2的其他核心元件相互作用以介导其底物分泌。通过细菌双杂交 (BACTH) 分析，我们现在表明 VgpA 与 VscN2（一种 T3SS 功能必需的 ATP 酶）和其他六种假设蛋白发生物理相互作用。 VgpA (VgpA ^I175A ) 残基 175 处的异亮氨酸突变为丙氨酸，消除了其与 VscN2 相互作用的能力。重要的是，VgpA 无义突变体 ( vgpA ') 与 VgpA ^I175A的互补并没有恢复 T3SS2 分泌底物的能力，这表明 VgpA-VscN2 相互作用对于 T3SS2 的功能至关重要。细菌细胞分级分离和质谱分析表明， vgpA '导致多个细菌细胞级分中T3SS2蛋白丰度的显着改变。特别地，与WT中的相比， vgpA '中内膜部分中的VscN2丰度和外膜部分中的VscC2丰度显着降低。这些结果表明，VgpA 通过与 VscN2 相互作用并可能通过影响 T3SS2 蛋白的亚细胞分布来促进 T3SS2 功能。