Denitrification genes regulate Brucella virulence in mice

Abstract

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. Genome sequencing of B. suis and B. melitensis revealed that both are complete denitrifiers. To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrification in the closely related B. neotomae was undertaken. In contrast to B. suis and B. melitensis, it was found that B. neotomae is a partial denitrifier that can reduce nitrate to nitrite but no further. Examination of the B. neotomae genome showed that a deletion in the denitrification gene cluster resulted in complete loss of nirV and the partial deletion of nirK and nnrA. Even though the nor operon is intact, a norC-lacZ promoter fusion was not expressed in B. neotomae. However, the norC-lacZ fusion was expressed in the related denitrifier Agrobacterium tumefaciens, suggesting that the lack of expression in B. neotomae is due to inactivation of NnrA. A narK-lacZ promoter fusion was found to exhibit nitrate-dependent expression consistent with the partial denitrifier phenotype. Complementation of the deleted region in B. neotomae by using nirK, nirV, and nnrA from B. melitensis restored the ability of B. neotomae to reduce nitrite. There was a significant difference in the death of IRF-1-/- mice when infected with B. neotomae containing nirK, nirV, and nnrA and those infected with wild-type B. neotomae. The wild-type strain killed all the infected mice, whereas most of the mice infected with B. neotomae containing nirK, nirV, and nnrA survived.

FIG. 1.

Comparison of the nirK-encoding regions of the B. suis and B. neotomae genomes. Arrows indicate locations of genes and their direction of transcription. Stippled arrows represent genes whose products are involved in denitrification. Vertical ticks mark distances of 1,000 bp. BRA0259 encodes a hypothetical protein, and its designation was assigned during genomic analysis of B. suis (24). (A) B. suis. The locations of the CGTGGC repeats are indicated. (B) B. neotomae. The region between the two CGTGGC sequences in panel A is not present, and the lone CGTGGC sequence in this region is indicated. nirK and nnrA are both partially deleted. The deletion removed the 3′ region of nirK, and so it is terminated by a line instead of an arrow. nnrA lacks its transcription start, and so it begins with a vertical mark.

FIG. 2.

CFU counts from livers and spleens of IRF-1^−/− mice infected with either B. neotomae or B. neotomae/pBgap. CFU counts were log transformed, and the data are an average from two mice at each time point. Material was plated on medium without any additional antibiotics. Diamonds, average liver CFU; squares, average CFU from spleens. Filled symbols are CFU from mice injected with wild-type B. neotomae; open symbols are CFU from mice injected with B. neotomae/pBgap. Error bars represent the range of CFU of the samples from each time point.