AN AROA MUTANT OF YERSINIA-PESTIS IS ATTENUATED IN GUINEA-PIGS, BUT VIRULENT IN MICE

This study describes a PCR-based approach for the production of a rati onally attenuated mutant of Yersinia pestis. Degenerate primers were u sed to amplify a fragment encoding 91.45% of the aroA gene of Y. pesti s MP6 which was cloned into pUC18. The remainder of the gene was isola ted by inverse PCR. The gene was sequenced and a restriction map was g enerated. The Y. pestis aroA gene had 75.9% identity with the aroA gen e of Yersinia enterocolitica. The cloned gene was inactivated in vitro and reintroduced into Y. pestis strain CB using the suicide vector pG P704. A stable are-defective mutant, Y. pestis GB Delta aroA, was isol ated and its virulence was examined in vivo. The mutant was attenuated in guinea-pigs and capable of inducing a protective immune response a gainst challenge with the virulent Y. pestis strain GB. Unusually for an are-defective mutant, the Y. pestis aroA mutant was virulent in mic e, with a median dose which induced morbidity or death similar to that of the wildtype, although time to death was significantly prolonged.