Type III secretion system (T3SS) activity is dependent on the EscV transmembrane sequences in Enteropathogenic Escherichia coli (EPEC)

Type III secretion system (T3SS) is highly sophisticated nanomachine that consists of approximately 20 different proteins that assemble to a syringe-like structure and facilitates delivery of many bacterial effectors directly from the cytoplasm of gram-negative bacteria into the host cells, thereby crossing the bacterial inner membrane, peptidoglycan and outer membrane, as well as the host plasma membrane. EscV protein, the main focus of our research, is one of the integral membrane proteins essential for the assembly and the function of T3SS. We found that at least three out of eight EscV predicted transmembrane domains (TMDs) mediate strong self-oligomerization in an isolated genetic reporter system. Replacing two transmembrane domains (TMD5 and TMD6) with an alternative hydrophobic sequence within the full-length protein eradicated substrate secretion and led to complete loss of function of the T3SS, including the ability to infect host cells in EPEC ΔescV mutant strains complemented with the TMD exchanged constructs. This suggests that EscV is not merely membrane anchor of the complex, but is required for the functioning of the T3SS. Moreover, we confirmed that replacing native EscV TMDs sequence with a hydrophobic one does not affect protein localization to the bacterial membrane. Finally, we examined interactions between EscV and other T3SS export apparatus components.