Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion

Enteropathogenic Escherichia coli (EPEC) secretes several Esps (E. coli-sec reted proteins) that are required for full virulence. Insertion of the bact erial protein Tir into the host epithelial cell membrane is facilitated by a type III secretion apparatus, and at least EspA and EspB are required for Tir translocation. An EPEC outer membrane protein, intimin, interacts with Tir on the host membrane to establish intimate attachment and formation of a pedestal-like structure. In this study, we identified a Tir chaperone, C esT, whose gene is located between tir and eae (which encodes intimin). A m utation in cesT abolished Tir secretion into culture supernatants and signi ficantly decreased the amount of Tir in the bacterial cytoplasm. In contras t, this mutation did not affect the secretion of the Esp proteins. The leve l of tir mRNA was not affected by the cesT mutation, indicating that CesT a cts at the post-transcriptional level. The cesT mutant could not induce hos t cytoskeletal rearrangements, and displayed the same phenotype as the fir mutant. Gel overlay and GST pulldown assays demonstrated that CesT specific ally interacts with Tir, but not with other Esp proteins. Furthermore, by u sing a series of Tir deletion derivatives, we determined that the CesT bind ing domain is located within the first 100 amino-terminal residues of Tir, and that the pool of Tir in the bacterial cytoplasm was greatly reduced whe n this domain was disrupted. Interestingly, this domain was not sufficient for Tir secretion, and at least the first 200 residues of Tir were required for efficient secretion. Gel filtration studies showed that Tir-CesT forms a large multimeric complex. Collectively, these results indicate that CesT is a Tir chaperone that may act as an anti-degradation factor by specifica lly binding to its amino-terminus, forming a multimeric stabilized complex.