Enteropathogenic Escherichia coli (EPEC) secretes several Esps (E. coli-secreted proteins) that are required for full virulence. Insertion of the bacterial 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, CesT, whose gene is located between tir and eae (which encodes intimin). A mutation in cesT abolished Tir secretion into culture supernatants and significantly decreased the amount of Tir in the bacterial cytoplasm. In contrast, this mutation did not affect the secretion of the Esp proteins. The level of tir mRNA was not affected by the cesT mutation, indicating that CesT acts at the post-transcriptional level. The cesT mutant could not induce host cytoskeletal rearrangements, and displayed the same phenotype as the tir mutant. Gel overlay and GST pulldown assays demonstrated that CesT specifically interacts with Tir, but not with other Esp proteins. Furthermore, by using a series of Tir deletion derivatives, we determined that the CesT binding 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 when 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 specifically binding to its amino-terminus, forming a multimeric stabilized complex.

To establish an intimate interaction with the host epithelial cell surface, enteropathogenic Escherichia coli (EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane constitutes the receptor for intimin. The tir gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by orf19 and downstream by the cesT and eae genes. With the use of a series of cat transcriptional fusions and primer extension analysis, we confirmed that tir, cesT, and eae form the LEE5 operon, which is under the control of a promoter located upstream from tir, and found that the orf19 gene is transcribed as a monocistronic unit. We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the tir and the orf19 promoters and that a sequence motif located between positions ؊204 and ؊157 was needed for the Ler-dependent activation of the tir operon. Sequence elements located between positions ؊204 and ؊97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions. Upon deletion of the upstream sequences, the tir promoter was fully active even in the absence of Ler, indicating that tir expression is subject to a repression mechanism that is counteracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25°C, suggesting that negative regulation also occurs at or downstream of the promoter. Expression of orf19, but not of the tir operon, became Ler independent in an hns mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histone-like nucleoid structuring protein) on this gene.Enteropathogenic Escherichia coli (EPEC) is a major cause of acute and persistent infantile diarrhea and a leading cause of infant death in developing countries (35,43,45). The interaction of EPEC with the host cell, which has been the subject of several recent reports, has been divided into three different stages characterized by two distinctive phenotypes, localized adherence and the attaching-and-effacing (A/E) lesion (reviewed in references 9, 11, 18, and 43). The virulence determinants required for the induction of the A/E lesion in EPEC are encoded in a 35.6-kb pathogenicity island, denoted LEE (for locus of enterocyte effacement), which contains 41 predicted open reading frames (16,38,39). Based on recent studies and sequence analyses, most of the LEE-encoded genes have been divided into three functional regions: the esc and sep genes, which code for a type III secretion-translocation apparatus (25); the tir, cesT, and eae genes, coding for the proteins involved in intimate attachment (1,14,26,28); and the esp genes, which encode effector proteins that are involved in the formation of a translocon for delivering effector molecules to the host cell (17,29,30,32,33,51).Recent studies have indicated that the LEE-encoded genes are organized into five major operons (14, 41): ...