DEVELOPMENT AND CHARACTERIZATION OF RECA MUTANTS OF CAMPYLOBACTER-JEJUNI FOR INCLUSION IN ATTENUATED VACCINES

Isogenic recA mutants of Campylobacter jejuni have been constructed fo r evaluation of their usefulness in attenuated vaccines against this m ajor worldwide cause of diarrhea. The recA(+) gene of C. jejuni 81-176 was cloned by using degenerate primers to conserved regions of other RecA proteins in a PCR. The C. jejuni recA(+) gene encodes a predicted protein with an M, of 37,012 with high sequence similarity to other R ecA proteins. The termination codon of the recA(+) gene overlaps with the initiation codon of another open reading frame which encodes a pre dicted protein which has >50% identity with the N terminus of the Esch erichia coli enolase protein. A kanamycin resistance gene was inserted into the cloned vecA(+) gene in E. coli and returned to C. jejuni VC8 3 by natural transformation, resulting in allelic replacement of the w ild-type recA gene. The resulting VC83 recA mutant displayed increased sensitivity to W light and a defect in generalized recombination as d etermined by natural transformation frequencies. The mutated recA gene was amplified from VC83 recA by PCR, and the product was used to tran sfer the mutation by natural transformation into C. jejuni 81-176 and 81-116, resulting in isogenic recA mutants with phenotypes similar to VC83 recA. After oral feeding, strain 81-176 recA colonized rabbits at levels comparable to wild-type 81-176 and was capable of eliciting th e same degree of protection as wild-type 81-176 against subsequent hom ologous challenge in the RITARD (removable intestinal tie adult rabbit diarrhea) model.