A directed intimin insertion mutant of a rabbit enteropathogenic Escherichia coli (REPEC) is attenuated, immunogenic and elicits serogroup specific protection

We previously showed (Agin et al., 2005) that a truncated beta-intimin mutant of an O15 A/E REPEC strain, which does not contain the C terminal tir binding region of intimin but expresses the preceding immunodominant portion of the molecule in outer membranes, is attenuated, induces anti-intimin and anti whole cell antibody, and protects against challenge with a virulent strain of the same serogroup. Since the ability of this and other intimin mutants lacking the tir binding region to provide broad protection against challenge with REPEC strains of other serogroups is incompletely studied, we generated a targeted insertion/deletion intimin mutant in an O103 strain, immunized rabbit's orogastrically, then challenged them with the parent strain and a virulent strain of a different serogroup. We used lambda red recombinase to generate an eae mutation in the prototypic rabbit A/E Escherichia coli strain E22 (O103) by replacing the 81 C-terminal (860-939) tir-binding amino acids of intimin with an inserted kanamycin resistance gene. This mutant did not express intimin in its outer membranes. A range of increasing immunizing doses (10(4)-10(7) CFU) was used for the first immunization in 4 groups of 6 rabbits. All 4 groups received a 2nd immunization with 10(7) CPU after 2 weeks. At 4 weeks, half of the rabbits in each group, and 6 control rabbits, were challenged with the parent O103 strain or with the O15 strain RDEC-H19A. All unimmunized rabbits exhibited characteristic weight loss with diarrhea and shedding of the challenge strain after challenge with E22 or RDEC-H19A. Rabbits challenged with the parent O103 E22, but not with the O15 RDEC-H19A, were protected against clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organism. At sacrifice, CPU of E22, but not RDECH19, were decreased in ileum, cecum and colon. Serum antibodies to E22 somatic antigens, but not intimin, were detected in rabbits immunized with E22 Delta eae(860-939) and correlated with protection. An intimin insertion mutation replacing the tir binding region of O103 REPEC strain E22 with an antibiotic resistance gene was attenuated, induced antibody to whole bacteria but not to intimin, and yielded protection against challenge with the WT strain from which it was prepared but not against a virulent strain of another serogroup. These results suggest that intimin expression in outer membranes may be necessary to confer cross serogroup protection by inducing anti-intimin immunity. Published by Elsevier B.V.