Brucella melitensis VjbR and C12-HSL regulons: contributions of the N-dodecanoyl homoserine lactone signaling molecule and LuxR homologue VjbR to gene expression

Abstract

Background: Quorum sensing is a communication system that regulates gene expression in response to population density and often regulates virulence determinants. Deletion of the luxR homologue vjbR highly attenuates intracellular survival of Brucella melitensis and has been interpreted to be an indication of a role for QS in Brucella infection. Confirmation for such a role was suggested, but not confirmed, by the demonstrated in vitro synthesis of an auto-inducer (AI) by Brucella cultures. In an effort to further delineate the role of VjbR to virulence and survival, gene expression under the control of VjbR and AI was characterized using microarray analysis.

Results: Analyses of wildtype B. melitensis and isogenic DeltavjbR transciptomes, grown in the presence and absence of exogenous N-dodecanoyl homoserine lactone (C12-HSL), revealed a temporal pattern of gene regulation with variances detected at exponential and stationary growth phases. Comparison of VjbR and C12-HSL transcriptomes indicated the shared regulation of 127 genes with all but 3 genes inversely regulated, suggesting that C12-HSL functions via VjbR in this case to reverse gene expression at these loci. Additional analysis using a DeltavjbR mutant revealed that AHL also altered gene expression in the absence of VjbR, up-regulating expression of 48 genes and a luxR homologue blxR 93-fold at stationary growth phase. Gene expression alterations include previously un-described adhesins, proteases, antibiotic and toxin resistance genes, stress survival aids, transporters, membrane biogenesis genes, amino acid metabolism and transport, transcriptional regulators, energy production genes, and the previously reported fliF and virB operons.

Conclusions: VjbR and C12-HSL regulate expression of a large and diverse number of genes. Many genes identified as virulence factors in other bacterial pathogens were found to be differently expressed, suggesting an important contribution to intracellular survival of Brucella. From these data, we conclude that VjbR and C12-HSL contribute to virulence and survival by regulating expression of virulence mechanisms and thus controlling the ability of the bacteria to survive within the host cell. A likely scenario occurs via QS, however, operation of such a mechanism remains to be demonstrated.

Figure 1

Intracellular survival of B. melitensis 16M (wt), vjbR mutant (ΔvjbR and vjbR::m Tn5), complemented ΔvjbR (ΔvjbRcomp and ΔvjbRvector), ΔblxR mutant, and 3 additional luxR-like mutants in J774A.1 murine macrophage-like cells. The attenuation was measured as the log difference between the CFU recoveries of the mutant compared to wildtype from infected macrophages at 48 hours post infection. Data shown is the averaged CFU recovery from at least 3 independent experiments, each performed in triplicate. Error bars represent the SEM and each mutant was compared to the wildtype by a Student's two tailed t-test, with the resulting p values as follows:*, P < .0.05; ***, P < 0.001. The luxR deletion mutant strains are identified by the BME gene locus ID tags, BME::Km representing the gene replacement mutant and ΔBME representing the gene deletion mutant.

Figure 2

Numbers and relationships of transcripts altered by the deletion of vjbR and/or treatment of C₁₂-HSL. Numbers represent the statistically significant transcripts found to be up or down-regulated by microarray analysis at the A) exponential growth phase (OD₆₀₀ = 0.4) and B) stationary growth phase (OD₆₀₀ = 1.5).

Figure 3

COG functional categories found to be over and under represented by the deletion of vjbR and the addition of C₁₂-HSL to wildtype cells, indicated by microarray analyses. Ratios were calculated by comparing the proportion of genes found to be altered by the putative QS component to the total number of genes classified in each COG category present in the B. melitensis genome.

Figure 4

Relative expression of vjbR transcript over time. Taqman real-time RT-PCR of vjbR in B. melitensis 16M expressed as the mean relative concentration (to 16srRNA) from 3 biological replicates ± standard deviation. Filled blue squares represent the relative expression of vjbR and the open light blue squares represent the OD₆₀₀ of corresponding cultures. The exponential growth stage for microarray analysis corresponds to OD₆₀₀ = 0.4 (14 hrs) and the stationary growth phase corresponds to OD₆₀₀ = 1.5 (28 hrs).