Characterization of the urease operon of Brucella abortus and assessment of its role in virulence of the bacterium

Abstract

Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

FIG. 1.

Optimum pH for urease activity. The urease activities of Brucella protein extracts were assayed in different buffer conditions with pHs ranging from 5.5 to 8. The experiment was performed three times in duplicate. Each point is the arithmetic mean of six independent experimental values. The error bars indicate standard deviations. Urease activity is expressed in micromoles of urea hydrolyzed per minute per milligram of protein.

FIG. 2.

Urease activity in the presence of ammonium chloride. B. abortus 2308 cultures were grown in BB in the presence of different amounts of added ammonium chloride, and their urease activities were measured. The activity is expressed as a percentage of the maximum activity that was obtained when no ammonium chloride was added to the medium. The experiment was performed in triplicate with three technical measurements per replicate. The bars indicate means, and the error bars indicate standard deviations.

FIG. 3.

Map of the ure1 and ure2 cluster region. The boxes represent the ORFs. The shaded boxes represent the structural genes, and the open boxes represent the accessory genes. The striped bars under ureC indicate the sequences deleted in mutants 2308ΔureC1 and 2308ΔureC2. The location of the Tn5 insertion in mutant BAM723 is indicated by an arrow.

FIG. 4.

Survival of B. abortus 2308 and urease mutants after acid exposure. Bacteria were resuspended in buffer at pH 4.0 (A) or in buffer at pH 2.0 (B) in the presence of different concentrations of urea (indicated at the bottom). After 30 min of incubation at 37°C, bacteria were diluted and plated to count the survivors. The data are the arithmetic means and standard deviations from three separate experiments. An unpaired t test was performed to determine if the survival of each strain was significantly different than the survival of the corresponding wild-type control. An asterisk indicates that the P value is <0.05. Light gray bars, BAM723; open bars, 2308ΔureC1; dark gray bars, 2308; solid bars, 2308ΔureC2.

FIG. 5.

Survival of the urease mutants after transit through the mouse stomach. A 1:1 mixture of 2308 and a urease mutant was administered orally to groups of five mice, and the experiment was performed twice. The bars indicate the competitive indexes (CI) for the three mutants with wild-type strain 2308 from one of the experiments. The error bars indicate standard deviations. The results of the mouse competitive index assay were analyzed with a nonparametric Mann-Whitney test using log-transformed competitive indexes to determine if the indexes were significantly different than 1. An asterisk indicates that the P value is <0.05. Gray bars, BAM723/2308; open bars, 2308ΔureC1/2308; solid bars, 2308ΔureC2/2308.