In vitro and in vivo phenotypes resulting from deletion of the high temperature requirement A (htrA) gene from the bovine vaccine strain Brucella abortus S19

An htrA deletion mutant was created in the bovine vaccine strain, B. abortus S19, by replacing the majority of the htrA gene with a kanamycin resistance gene. Antibiotic selection for a double crossover event yielded kanamycin-resistant, ampicillin-sensitive colonies confirmed by Southern and western blot analysis to be HtrA deficient. The B. abortus S19 htrA mutant was significantly more susceptible than the parental strain to killing by H2O2 (P \u3c 0.001) and O2- generated by the redox cycling agent paraquat (P \u3c 0.05) in disk sensitivity assays. Deletion of the htrA gene from S19 produced a bimodal effect on the spleen colonization profile of this strain in BALB/c mice. At one week post-infection, the B. abortus S19 htrA mutant colonized the spleens of experimentally infected BALB/c mice at significantly lower levels (P \u3c 0.01) than the parental strain. Enhanced clearance (P \u3c 0.05) was also observed at later time points, i.e. 4 and 7 weeks post infection, however at 2 and 3 weeks post infection, the mutant and parental strains colonized the mice at equivalent levels. The temporal development of specific delayed type hypersensitivity and antibody responses in BALB/c mice infected with the mutant or parental strain were equivalent. These results suggest that the htrA gene product contributes to successful host colonization by S19. However, deletion of this gene does not radically alter the overall, characteristic spleen colonization profile of this vaccine strain in the BALB/c mouse model, nor compromise the capacity of this strain to elicit Brucella specific cellular or humoral immune responses in this experimental host