Role of Tir and Intimin in the Virulence of Rabbit EnteropathogenicEscherichia coliSerotype O103:H2

Abstract

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.Although Escherichia coli belongs to the normal microflora present in the gastrointestinal tracts of most mammals and birds, certain E. coli strains have been associated with intestinal or extraintestinal infections. Among these pathogenic E. coli strains, enteropathogenic E. coli (EPEC) is a major cause of infant diarrhea in developing countries (for a recent review, see reference 51) and is a significant category of diarrheagenic E. coli in different animal species. In addition, EPEC is an important cause of morbidity and mortality in weaned rabbits (5,54,56). EPEC is also pathogenic in neonatal calves (20,53) and seems to be isolated more frequently in farms with recurrent diarrhea (7). In swine, EPEC is involved in cases of postweaning diarrhea (67). There is also increasing evidence for a diarrheagenic role of EPEC in dogs (16,64). Finally, EPEC has been isolated ...

Hierarchical Delivery of an Essential Host Colonization Factor in Enteropathogenic Escherichia coli

“…EPEC tir mutants do not cause diarrhea in weaned rabbits and do not intimately adhere to host intestinal cells (17).…”

“…Remarkably, host membrane-localized Tir also serves as a receptor for EPEC via intimin (10), a bacterial outer membrane adhesin also encoded by the LEE of A/E pathogens. The direct importance of Tir in the virulence of A/E pathogens has been convincingly demonstrated in three in vivo animal models of infection, where tir mutants do not colonize the host intestine or cause clinical symptoms of disease (7,17,18). Other well studied LEE encoded type III effectors include Map, EspF, and EspG (19 -22), which have been shown to have multifunctional disruptive properties within host cells.…”

In Vitro and In Vivo Model Systems for Studying Enteropathogenic Escherichia coli Infections

“…Similar to human-specific EPEC strains, REPEC induces A/E lesions and intestinal pathology without invasion of the epithelia or production of enterotoxins (RobinsBrowne et al 1994b). The REPEC LEE has been characterized and shares a high degree of homology with the LEE region of EPEC (Tauschek et al 2002), thereby facilitating the discovery of LEE genes required for A/E lesion formation and disease (Abe et al 1998;Marchès et al 2000).…”

“…BFP is an EPEC-specific virulence factor but intimin and EspB are found in the genomes of diverse A/E pathogen species (Tauschek et al 2002;Iguchi et al 2009;Ogura et al 2009;Petty et al 2010). Intimin and EspB are essential for A/E lesion formation in tissue culture (Foubister et al 1994;Abe et al 1997) and intestinal damage in rabbits and mice infected with REPEC (Abe et al 1998;Marchès et al 2000) or C. rodentium, respectively (Schauer and Falkow 1993;Newman et al 1999;Deng et al 2004). Intestinal biopsies taken from human volunteers infected with wild-type EPEC show destruction of the epithelial brush border (Tacket et al 2000), a phenotype that is reproducible in tissue culture (Knutton et al 1987).…”

Options for the control of enterohaemorrhagic Escherichia coli in ruminants

“…The site(s) of specific adherence of E. coli O157 : H7 to the intestinal epithelium in calves remain to be determined. LEE-encoded genes are also crucial for colonization of the rabbit intestine by rabbit EPEC (Abe et al, 1998 ;Marche! s et al, 2000).…”