A gatekeeper protein contributes to T3SS2 function via interaction with an ATPase in Vibrio parahaemolyticus

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.