Enterohaemorrhagic Escherichia coli O157 : H7 is a human pathogen that causes no apparent disease in cattle, its primary reservoir host. Recent research has demonstrated that E. coli O157 : H7 predominately colonizes the distal few centimetres of the bovine rectum, and in this study, the LEE4 operon encoding a type III secretion system translocon and associated proteins was shown to be essential for colonization. A deletion mutant of LEE4 failed to colonize cattle, in contrast to a co-inoculated strain containing a chromosomal complement of the operon, therefore fulfilling 'molecular' Koch's postulates for this virulence determinant. In addition, attaching and effacing (A/E) lesions were detectable in E. coli O157 : H7 microcolonies from the terminal rectum of both naturally and experimentally colonized cattle when examined by transmission electron microscopy. This study proves that type III secretion is required for colonization of cattle by E. coli O157 : H7, and that A/E lesion formation occurs at the bovine terminal rectum within E. coli O157 : H7 microcolonies. The research confirms the value of using type III secreted proteins as vaccine candidates in cattle. INTRODUCTIONEnterohaemorrhagic Escherichia coli (EHEC) has emerged in developed countries over the past 20 years as an important cause of human intestinal disease. In addition to bloody diarrhoea, intestinal infection can lead to potentially fatal systemic sequelae resulting from the activity of Shiga toxins. The majority of these infections in the USA, Canada, UK and Japan are caused by E. coli O157 : H7 (Nataro & Kaper, 1998). This serotype has been frequently isolated from cattle faeces, and most human E. coli O157 : H7 infections originate, either directly or indirectly, from this source (Besser et al., 1999;Borczyk et al., 1987). It is widely acknowledged that controlling E. coli O157 : H7 within the bovine population would be an effective method of reducing transmission to humans (Stevens et al., 2002).In common with other EHEC and EPEC (enteropathogenic E. coli), E. coli O157 : H7 contains a pathogenicity island, known as the locus of enterocyte effacement (LEE), that confers the attaching and effacing (A/E) phenotype McDaniel & Kaper, 1997). The LEE encodes a type III secretion system (TTSS) (Hueck, 1998), various translocators and effectors, the outer-membrane protein intimin (Jerse et al., 1990) and its receptor, termed Tir (translocated intimin receptor) (Kenny et al., 1997). The LEE is arranged into several polycistronic operons termed LEE1 to LEE5 (Elliott et al., 1998). The LEE4 operon encodes several proteins essential for the A/E phenotype. These include SepL (Kresse et al., 2000) and EscF (Wilson et al., 2001), both essential components of the LEE TTSS, and Esps (EPEC secreted proteins) A, B, D and F (Knutton et al., 1998;Taylor et al., 1998;Wachter et al., 1999; and the non-LEE-encoded Cif, EspI/NleA and TccP (Marches et al., 2003;Mundy et al., 2004;Gruenheid et al., 2004;Garmendia et al., 2004). Other than EspF, these are all likel...

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This review covers enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infections, focusing on differences in their virulence factors and regulation. While Shiga-toxin expression from integrated bacteriophages sets EHEC apart from EPEC, EHEC infections often originate from asymptomatic carriage in ruminants whereas human EPEC are considered to be overt pathogens and more host-restricted. In part, these differences reflect variation in adhesin repertoire, type III-secreted effectors and the way in which these factors are regulated.

Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a serotype O26:H-strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 causes gastrointestinal disease in humans that can be life threatening as a consequence of Shiga toxin activity on kidney and brain vasculature. Ruminants, particularly cattle, are the primary reservoir hosts for this organism (1, 3). Cattle are colonized without any overt symptoms and can shed EHEC O157:H7 in their feces for several weeks at levels up to 10 6 cells per g. Recent work has demonstrated that the predominant colonization site in cattle for E. coli O157:H7 is the final few centimeters of the terminal rectum, a site having a high density of lymphoid follicles and lymphoid-associated mucosa (19,21,30). A key aim of our research is to identify bacterial factors that drive this tropism for the terminal rectum.Considerable attention has been focused on type III secretion that leads to intimate attachment, and this has been shown to be important for ruminant colonization in a number of studies (5,7,8,22,44). For example, in a recent signaturetagged mutagenesis (STM) screening for genes that promote E. col...