Role of hha and ler in Transcriptional Regulation of the esp Operon of Enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a Shiga toxin-producing E. coli, causes a broad spectrum of diseases, including uncomplicated diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (21). In addition to Shiga toxins, which act on vascular endothelial cells to produce hemolytic uremic syndrome, EHEC O157:H7 harbors genes mediating its adherence to intestinal epithelial cells by a characteristic attaching-and-effacing mechanism (31). The attaching and effacing lesions are characterized by the loss of microvilli and the formation of pedestals of polymerized actin and other cytoskeletal elements underneath and around the infecting bacterial cells (10). The attaching and effacing phenotype requires concerted action of several genes contained within a pathogenicity island, called the locus of enterocyte effacement (LEE) (32). LEE encodes a type III secretion system, an outer membrane protein called intimin, a translocated intimin receptor called Tir, and secreted proteins EspA, EspD, and EspB. The genes espA, espD, and espB, which encode Esp proteins, are organized into a single operon (2) and are secreted by the type III secretion apparatus. EspA forms a novel filament-like structure which is essential for early bacterial attachment to epithelial cells and translocation of EspB and Tir into host cells (11, 22). Nucleotide sequence analysis of the LEE region of EHEC O157:H7 strain EDL 933 (37) revealed the presence of 41 open reading frames (ORFs), most of which are organized into five polycistronic operons named LEE1 through LEE4 and tir. The genetic organization of the LEE-encoded ORFs of EHEC O157:H7 is similar to that reported for the LEE of enteropathogenic E. coli (EPEC) O127:H6 (13). The Ler protein, encoded by the gene ler of the LEE1 operon, is essential for transcriptional activation of the LEE2, LEE3, and tir operons, which encode many of the protein factors required for the production of the attaching and effacing phenotype by EHEC and EPEC strains on host cells (12). Ler has also been shown to be a weak regulator of esp operon (LEE4) expression in EPEC (28). Ler shows some similarities with H-NS (histone-like nucleoid-structuring protein) and StpA (suppressor of td− phenotype A) of Salmonella enterica and E. coli, predominantly with the DNA-binding domain near the carboxy terminus (3, 13). Binding of Ler to the LEE2 regulatory region is required for activation of both the LEE2 and LEE3 operons (43), and Ler counteracts the negative regulation exerted by some H-NS proteins on the expression of LEE2 and LEE3 in EPEC (6). However, the genetic factors required for expression of the esp operon in EHEC O157:H7 are not fully understood. The objective of this study was to identify transcriptional regulators of esp (LEE4). Our approach was to use transposon mutagenesis of EHEC O157:H7 strain 86-24 Δstx2 Δlac carrying an esp::lac transcriptional fusion to identify mutants with altered β-galactosidase activity compared to the parent strain and then characterize the genes altered by the transposon insertions. With this strategy, hha was identified as an indirect negative regulator of esp. The gene hha is implicated in the negative regulation of α-hemolysin expression in pathogenic E. coli (33). We provide several lines of data that show that Hha represses ler transcription and Ler is a positive regulator of esp expression. (A preliminary account of this work was presented at the 102nd Annual Meeting of the American Society for Microbiology, Salt Lake City, Utah, 19 to 23 May 2002 [V. K. Sharma and R. W. Morgan, Abstr. 102nd Annu. Meet. Am. Soc. Microbiol., abstr. B269, p.78, 2002].)