Global analysis of quorum sensing targets in the intracellular pathogen Brucella melitensis 16 M

Abstract

Many pathogenic bacteria use a regulatory process termed quorum sensing (QS) to produce and detect small diffusible molecules to synchronize gene expression within a population. In Gram-negative bacteria, the detection of, and response to, these molecules depends on transcriptional regulators belonging to the LuxR family. Such a system has been discovered in the intracellular pathogen Brucella melitensis, a Gram-negative bacterium responsible for brucellosis, a worldwide zoonosis that remains a serious public health concern in countries were the disease is endemic. Genes encoding two LuxR-type regulators, VjbR and BabR, have been identified in the genome of B. melitensis 16 M. A DeltavjbR mutant is highly attenuated in all experimental models of infection tested, suggesting a crucial role for QS in the virulence of Brucella. At present, no function has been attributed to BabR. The experiments described in this report indicate that 5% of the genes in the B. melitensis 16 M genome are regulated by VjbR and/or BabR, suggesting that QS is a global regulatory system in this bacterium. The overlap between BabR and VjbR targets suggest a cross-talk between these two regulators. Our results also demonstrate that VjbR and BabR regulate many genes and/or proteins involved in stress response, metabolism, and virulence, including those potentially involved in the adaptation of Brucella to the oxidative, pH, and nutritional stresses encountered within the host. These findings highlight the involvement of QS as a major regulatory system in Brucella and lead us to suggest that this regulatory system could participate in the spatial and sequential adaptation of Brucella strains to the host environment.

Figure 1

Diagram representing the main metabolic pathways in the wt strain and the regulation effect of VjbR and BabR. Pentose-P, pentose phosphate pathway; TCA, tricarboxylic acid cycle; G1P, glycerol-1-P; F6P, fructose-6-P; Ga3P, glyceraldehyde-3-P; 3PG, 3-P-glycerate; PYR, pyruvate; OXA, oxaloacetate; ISO, isocitrate; SUC; succinate; GLU, glutamate; Bile salt, glycocholate or taurocholate. Red lines/arrows represent repressed pathways while green lines/arrows represent activated pathways by the regulator. a.a., amino acid.

Figure 2

wt, ΔvjbR and ΔbabR resistance to bile salts. Strains were growth in 2YT with bile salts and CFU were compared with cultures in 2YT (100% of survival). Error bars represent standard deviation from three independent experiments. CFU, colony forming unit.

Figure 3

B. melitensis wt, ΔvjbR and ΔbabR response to acid and alkaline stresses. Strains were growth in 2YT pH 5, 7, or 9 and CFU were compared with cultures in 2YT (100% of survival). Error bars represent standard deviation from three independent experiments. CFU, colony forming unit.

Figure 4

ChIP experiments showing direct binding of VjbR on several promoter regions. (A) Detection of several virB1-virB2 regions. (B) Detection of BMEII0734, BMEII0590, BMEI0030, BMEI1305, BMEI0668 and BMEI1007 promoter regions. All ChIP were performed with a C-terminal Flag-tagged VjbR-HTH protein expressed from a high copy plasmid (pSB502). The y-axis represents the ratio of immunoprecipited product (IP) versus input (IN) (%IP/IN). White columns represent IP from control strain (ΔvjbR, empty plasmid), gray columns represent IP from (ΔvjbR, pSB502). Error bars represent standard deviation from three independent experiments.