Quorum sensing and self-quorum quenching in the intracellular pathogen Brucellamelitensis.

Brucella quorum sensing has been described as an important regulatory system controlling crucial virulence determinants such as the VirB type IV secretion system and the flagellar genes. However, the basis of quorum sensing, namely the production of autoinducers in Brucella has been questioned. Here, we report data obtained from the use of a genetic tool allowing the in situ detection of long-chain N-acyl-homoserine lactones (AHL) activity at single bacterium level in Brucella melitensis. These data are consistent with an intrinsic production of AHL by B. melitensis in low concentration both during in vitro growth and macrophage infection. Moreover, we identified a protein, named AibP, which is homologous to the AHL-acylases of various bacterial species. In vitro and during infection, expression of aibP coincided with a decrease in endogenous AHL activity within B. melitensis, suggesting that AibP could efficiently impair AHL accumulation. Furthermore, we showed that deletion of aibP in B. melitensis resulted in enhanced virB genes expression and VirB8 production as well as in a reduced flagellar genes expression and production of FlgE (hook protein) and FliC (flagellin) in vitro. Altogether, these results suggest that AHL-dependent quorum sensing and AHL-quorum quenching coexist in Brucella, at least to regulate its virulence.

The two-component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength.

Bacterial adaptation to environmental conditions is essential to ensure maximal fitness in the face of several stresses. In this context, two-component systems (TCSs) represent a predominant signal transduction mechanism, allowing an appropriate response to be mounted when a stimulus is sensed. As facultative intracellular pathogens, Brucella spp. face various environmental conditions, and an adequate response is required for a successful infection process. Recently, bioinformatic analysis of Brucella genomes predicted a set of 15 bona fide TCS pairs, among which some have been previously investigated. In this report, we characterized a new TCS locus called prlS/R, for probable proline sensor-regulator. It encodes a hybrid histidine kinase (PrlS) with an unusual Na(+)/solute symporter N-terminal domain and a transcriptional regulator (belonging to the LuxR family) (PrlR). In vitro, Brucella spp. with a functional PrlR/S system form bacterial aggregates, which seems to be an adaptive response to a hypersaline environment, while a prlS/R mutant does not. We identified ionic strength as a possible signal sensed by this TCS. Finally, this work correlates the absence of a functional PrlR/S system with the lack of hypersaline-induced aggregation in particular marine Brucella spp.

A Brucella abortus cstA mutant is defective for association with endoplasmic reticulum exit sites and displays altered trafficking in HeLa cells.

Members of the genus Brucella are facultative intracellular pathogenic bacteria able to control maturation of their vacuoles. In several cell types, Brucella is able to reach a proliferation compartment derived from the endoplasmic reticulum (ER). Since ER exit site (ERES) functions are required for Brucella proliferation, we performed a yeast two-hybrid screen between human ERES-associated proteins and the predicted brucella proteome. This screening led to the identification of CstA, a conserved protein that specifically interacts with Sec24A, a component of the ERES. We found that a tagged CstA is secreted in Brucella abortus culture medium. This secretion is independent of the type IV secretion system VirB and the flagellum, suggesting that CstA is secreted through another system. We also discovered that a B. abortus cstA mutant is impaired for its association with the Sec23 ERES marker. The B. abortus cstA mutant displayed peculiar trafficking, with reduced association with LAMP1 and Calnexin 12 h post-infection in HeLa cells. However, its intracellular proliferation kinetics was not affected. The data reported here suggest that CstA could be directly or indirectly involved in the control of B. abortus intracellular trafficking in HeLa cells.

Brucella melitensis 16M produces a mannan and other extracellular matrix components typical of a biofilm.

Mutations in the Brucella melitensis quorum-sensing (QS) system are involved in the formation of clumps containing an exopolysaccharide. Here, we show that the overexpression of a gene called aiiD in B. melitensis gives rise to a similar clumping phenotype. The AiiD enzyme degrades AHL molecules and leads therefore to a QS-deficient strain. We demonstrated the presence of exopolysaccharide and DNA, two classical components of extracellular matrices, in clumps produced by this strain. We also observed that the production of outer membrane vesicles is strongly increased in the aiiD-overexpressing strain. Moreover, this strain allowed us to purify the exopolysaccharide and to obtain its composition and the first structural information on the complex exopolysaccharide produced by B. melitensis 16M, which was found to have a molecular weight of about 16 kDa and to be composed of glucosamine, glucose and mostly mannose. In addition, we found the presence of 2- and/or 6-substituted mannosyl residues, which provide the first insights into the linkages involved in this polymer. We used a classical biofilm attachment assay and an HeLa cell infection model to demonstrate that the clumping strain is more adherent to polystyrene plates and to HeLa cell surfaces than the wild-type one. Taken together, these data reinforce the evidence that B. melitensis could form biofilms in its lifecycle.