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Shiga toxin-producing strains of enterohemorrhagic Escherichia coli (EHEC) are a prominent cause of acute gastroenteritis, hemorrhagic colitis, and the hemolytic-uremic syndrome (HUS) in humans (17, 23). HUS is characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia, and those most at risk of developing renal disease are children under 10 years and the elderly. In approximately 3 to 5% of affected children, HUS is fatal, while 12 to 30% suffer serious impairment of renal function or neurologic damage (17, 23). Although the carriage of bacteriophage-encoded Shiga toxin genes (stx) is a defining feature of virulence, strains of EHEC also harbor a large ∼90-kb plasmid, pEHEC, and/or a large chromosomal pathogenicity island, termed the locus for enterocyte effacement (LEE) (6, 14). Strains of EHEC carrying LEE belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-effacing (A/E) lesions. This group of organisms also includes the human pathogen enteropathogenic E. coli (EPEC) and the animal pathogens rabbit-specific enteropathogenic E. coli (REPEC) and Citrobacter rodentium. A/E lesions are characterized by localized destruction (effacement) of intestinal brush border microvilli, intimate attachment of the bacteria to the host cell membrane, and the formation of underlying actin-rich, pedestal-like structures in the host cell (11, 17). The formation of A/E lesions is essential for bacterial colonization of the intestinal mucosa by LEE-positive pathogens. Knockout mutations in eae, espB, tir, and espA abolish the ability of A/E pathogens to cause A/E lesions and seriously impair the capacity of these organisms to colonize the host and cause disease (1, 8, 15, 16, 28, 31). Nevertheless, some serotypes of EHEC do not carry LEE and are not A/E pathogens (9, 22). These strains have regularly been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS, but little is known about the way in which these LEE-negative EHEC adhere to and colonize the human intestine. Clinical isolates of LEE-negative EHEC typically express Shiga toxin type 2 and also harbor an ∼90-kb plasmid which encodes EHEC hemolysin (22). However, restriction fragment length polymorphism analysis of the ehxA gene has shown that the large plasmids from LEE-negative EHEC comprise an evolutionarily distinct group compared with the similarly sized plasmids of LEE-positive EHEC (5). EHEC O113:H21 is one of the most commonly isolated LEE-negative EHEC serotypes worldwide, but until recently no virulence-associated genes apart from stx and ehx in these bacteria had been documented. Recently, however, Paton et al. identified a novel plasmid-encoded autoagglutinating adhesin, termed Saa, in LEE-negative EHEC O113:H21 (21). Saa is a 516-amino-acid protein with a low degree of similarity to the adhesin YadA of Yersinia enterocolitica. Mutation of the saa gene or curing of the pO113 plasmid resulted in reduced adhesion to epithelial cells (21). Dytoc et al. have demonstrated the pili on the surfaces of EHEC O113:H21 cells, but the genes coding for pili in these strains or any LEE-negative EHEC have not been described (9). The most common serotype of EHEC associated with outbreaks and sporadic disease worldwide is O157:H7 (14). Recently, the genome sequences of two EHEC O157:H7 outbreak strains were completed. Analysis of these sequences showed that EHEC O157:H7 has 1.34 Mb of DNA that is not present in nonpathogenic E. coli K-12 (12, 24). The additional regions of DNA (termed O islands) are inserted into a common “backbone” shared by E. coli K-12 and EHEC O157:H7 and vary in size from a few hundred base pairs up to 88 kb. About 33% of the 177 O islands contain only genes of unknown function. Many genes in the other O islands have been classified according to their homology with known virulence factors, although their role in disease is not proven (24). Several O islands contain putative fimbrial biosynthesis operons, including two regions predicted to encode long polar fimbriae (LPF). LPF are related to type I fimbriae in genetic organization and were first identified in Salmonella enterica serovar Typhimurium (3). They form surface structures 7 to 8 nm in diameter and 2 to 10 μm in length extending from the poles of the bacterial cell. In S. enterica serovar Typhimurium, LPF have been shown to facilitate attachment of the bacteria to murine Peyer's patches. Synthesis of LPF is controlled by a phase-variable on/off switch that regulates expression of LPF in different tissues and organs (4). Phase variation of LPF is also a mechanism by which S. enterica serovar Typhimurium evades cross immunity among different Salmonella serotypes (19). In EHEC O157:H7, two putative lpf operons are present as O islands 141 and 154; however, the expression and production of LPF in EHEC O157:H7 has not yet been demonstrated, and their contribution to virulence is unknown (24). In this study, we report the identification of a novel fimbrial gene cluster related to LPF in LEE-negative EHEC serotype O113:H21. We also describe the contribution of these genes to the adherence of EHEC O113:H21 to epithelial cells and the relationship of lpfO113 to lpf gene clusters in other bacteria.

Enteropathogenic E. coli(EPEC) is an important diarrhoeal pathogen that induces characteristic lesions on the host intestine termed attaching and effacing (A/E) lesions. In this study we have examined the contribution of a large gene, efa1, which is present in all A/E pathogens, to the adherence phenotype of EPEC. An efa- derivative of EPEC JPN15 was constructed and this mutant was significantly less adherent to epithelial cells than the parent strain. The JPN15 efa- derivative was FAS-positive, produced EspA filaments and showed comparable levels of EspA secretion to JPN15. In addition, polyclonal antibodies raised to Efa1 partially inhibited the adherence of JPN15 to cultured epithelial cells. In further work, we showed that human and rabbit hosts infected with an A/E pathogen produced antibodies to Efa1 and we observed that the truncated form of efa1 present in EHEC O157:H7 was specific to that serotype. Generally efa1 was present in its entirety in the genomes of other A/E pathogens. Overall our data suggest that Efa1 has host cell binding activity, at least in tissue culture, and that it is produced during infection. These findings suggest that Efa1 may play a direct role in the pathogenesis of infections caused by A/E pathogens.

Colonisation of host tissue by Gram- negative bacteria is facilitated by various adhesins, one of which is type 4 fimbriae (pili). These structures have been associated with pathogenesis in several bacterial species, and have been shown to mediate colonisation of epithelial surfaces. Recently, type 4 fimbriae were identified and characterised from P. multocida strains A, B and D. The type 4 fimbrial subunit protein (PtfA) was identified as an 18-kDa protein which was isolated from whole membrane fractions. We report here the isolation and characterisation of the gene (ptfA) encoding the PtfA protein from P. multocida VP161 (serotype A:1). Part of the gene was cloned on a 2-kb genomic DNA fragment. The complete ptfA gene was obtained using inverse PCR. The gene and its flanking regions were characterised, and the deduced PtfA amino acid sequence was compared to type 4 subunit protein sequences from other bacterial species. The ptfA gene was amplified and sequenced from several P. multocida strains. Comparison of these sequences revealed variation within the type 4 subunit gene of P. multocida.

Pasteurella multocida is the causative agent of fowl cholera and other diseases of production animals. Isolates are classified into five groups based on capsular antigens and into 16 serotypes based on LPS antigens. Strains causing fowl cholera are most frequently designated A:1, A:3 or A:4. Whole cell bacterins can provide some degree of protection, but only against the homologous LPS serotype. There is good evidence that cross-protective antigens are expressed only under in vivo conditions. Empirically derived, live, attenuated vaccines can protect against heterologous serotypes, but because the basis for attenuation is undefined, reversion to virulence is not uncommon. Work in our laboratory is aimed at using a variety of approaches to identify potential protective antigens or virulence genes to be used as candidates for attenuating mutations or as the basis for vaccine antigen delivery systems. The gene encoding an outer membrane protein, Oma87, which is a homologue of the D15 protective antigen of Haemophilus influenzae, was cloned and sequenced. Rabbit antiserum prepared against recombinant Oma87 could passively protect mice against infection. Type 4 fimbriae form the basis of vaccines against ovine footrot and bovine keratoconjunctivitis. We have identified type 4 fimbriae on the surface of P. multocida, purified the fimbrial subunit protein, PtfA, and determined its N-terminal amino acid sequence. Subsequent cloning of the ptfA gene and its inactivation will now be used to assess the importance of type 4 fimbriae in virulence. There has long been anecdotal evidence for the importance of capsule in virulence, but unequivocal genetic evidence for such a role is lacking. We have cloned and characterised the capsule biosynthetic locus in P. multocida A:1 and identified four bex genes involved in capsule transport and genes encoding enzymes involved in the biosynthesis and transfer of the N-acetyl glucosamine and glucuronic acid components of the capsule. It has been suggested that the low concentration of available iron in vivo acts as an environmental cue for expression of cross-protective antigens. Accordingly, we have cloned and characterised the gene encoding transferrin binding protein, Tbpl, so that its role in immunity and virulence can be investigated. Although P. multocida is not normally considered haemolytic, we have observed haemolysis under anaerobic conditions. Standard library construction and screening resulted in the identification of the mesA gene which encodes an esterase enzyme resulting in a haemolytic phenotype under anaerobic conditions. Virulence studies with mesA- mutants were performed to assess its role in pathogenesis. Using a promoterless phoA gene vector system, the cloning of proteins homologous to known surface proteins of other species as well as proteins unique to P. multocida, allowing their potential as vaccine components to be assessed.