Characterization and Protective Property of Brucella abortuscydCandlooPMutants
ABSTRACTBrucella abortusreadily multiplies in professional or nonprofessional phagocytesin vitroand is highly virulent in mice. Isogenic mutants ofB. abortusbiovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (namedlooP; designated here the IVKB9007looP::Tn5mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007cydC::Tn5mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007looP::Tn5and IVKB9007cydC::Tn5mutants restored their ability to survivein vitroandin vivoto a level comparable with that of the wild type. These findings indicate that thecydCandlooPgenes play important roles in the virulence ofB. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007looP::Tn5and IVKB9007cydC::Tn5mutants provided a high degree of protection against challenge with pathogenicB. abortusstrain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response.