The transmembrane domains of secreted proteins are involved in host-pathogen interactions

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen and a major cause of gastrointestinal illness in developing countries. EPEC possess an essential virulence machinery called Type III Secretion System (T3SS) that enable the injection of numerous effector proteins from the bacteria into the host cytoplasm. The translocated Intimin receptor (Tir) is the first effector to be injected and its activity is essential for the hallmark of EPEC colonization of the intestinal mucosa – "attaching and effacing" (A/E) lesions. Tir belongs to a unique group of transmembrane (TMD) containing secreted proteins, which have contradicting indications for both membranal integration and secretion by the T3SS. Here, we characterized the roles of Tir TMDs in the protein function. For this purpose, we created Tir variants where the TMD sequences were replaced by the other Tir natural TMD or with an alternative TMD sequence. Our results demonstrated that the TMD sequence is important for the determination of the protein fate – bacterial membrane integration or secretion and suggest that the second TMD of Tir is critical for the ability of Tir to avoid integration into the bacterial membrane. We also found that the second TMD of Tir is important for its post-secretion function, at the host cell, in a yet to be discovered mechanism. Additionally, we observed that the context of the TMD within the whole protein sequence affect protein secretion. Taken together, our study further supports the hypothesis that the TMD sequences of translocated proteins encode information crucial for protein secretion and function.