Role of Tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103 : H2

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPE C) strains belonging to serogroup O103 are an important cause of diarrhea i n weaned rabbits. Like human EPEC strains, they possess the locus of entero cyte effacement clustering the genes involved in the formation of the A/E l esions, In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton cha racterized by the formation of focal adhesion complexes and the reorganizat ion of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogeni city of REPEC, we have used a newly constructed isogenic fir null mutant to gether with a previously described eae null mutant. When human HeLa epithel ial cells were infected, the tir mutant was still able to induce the format ion of stress fibers as previously reported for the eae null mutant. When t he rabbit epithelial cell line RK13 was used, REPEC O103 produced a classic al fluorescent actin staining (FAS) effect, whereas both the eae and tir mu tants were FAS negative. In a rabbit ligated ileal loop model, neither muta nt was able to induce A/E lesions. In contrast to the parental strain, whic h intimately adhered to the enterocytes and destroyed the brush border micr ovilli, bacteria of both mutants were clustered in the mucus without reachi ng and damaging the microvilli. The role of intimin and Tir was then analyz ed in vivo by oral inoculation of weaned rabbits. Although both mutants wer e still present in the intestinal flora of the rabbits 3 weeks after oral i noculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused t he death of 90% of the inoculated rabbits. Nevertheless, an inflammatory in filtrate was present in the lamina propria of the rabbits infected with bot h mutants, with an inflammatory response greater for the eae null mutant, I n conclusion, we have confirmed the role of intimin in virulence, and we ha ve shown, for the first time, that Tir is also a key factor in vivo for pat hogenicity.