Role of the transcriptional regulator RegA in establishment of Brucella suis persistence in an original in vitro model.
Résumé
Low oxygen tension is one of the environmental conditions encountered by Brucella during intramacrophagic replication and chronic infection of the host. At chronic stage of brucellosis, these bacteria can be found in granulomas that tend to evolve in abscesses where anoxic conditions predominate. Therefore, tackling the capacity of this facultative aerobic microorganism to adapt and persist under oxygen deficiency is of major interest. In the past few years, we demonstrated the high metabolic flexibility of Brucella suis with respect to oxygen deprivation (micro- and anaerobiosis). We evidenced the central role of the two-component system RegB/RegA in the coordinated control of oxidative respiration and denitrification respiratory systems, which are crucial for virulence and/or persistence in vivo. More importantly, RegA was found to be essential for B. suis persistence in mice. Recently, we developed an original in vitro model, characterized by progressive oxygen deprivation in minimal medium, which allowed to show that RegA is essential for optimal long-lasting in vitro persistence. To identify genes and proteins RegA-dependent in this model, global transcriptomic analysis (microarrays) and whole proteome quantifications (2-D DIGE) were performed by comparison of the wild-type B. suis with a regA mutant strain. These analyses were performed at the time point where anaerobic conditions become established, which corresponded to the cessation of wild-type strain multiplication. Genetic validation by quantitative PCR (RT-qPCR) indicated that RegA potentially regulates 12% of the B. suis genes and its role in bacterial adaptation to oxygen deficiency was confirmed.The down-regulation of genes or proteins involved in cell envelope biogenesis and in cellular division suggests that RegA could be involved in establishment of a non-replicative state. In addition, RegA-dependent repression of an important number of genes involved in energy production could be indicative of a participation of RegA in the slowing-down of central metabolim as it enters into the persistence phase. This was substantiated by the finding that two-thirds of the differentially produced proteins belonging to this functional class were also found repressed. Notably, among them was detected the isocitrate lyase, the first enzyme of the glyoxylate shunt, encoded by the most down-regulated gene. Several genes of the virB operon were also found repressed by RegA as was its regulator VjbR. In addition, biological validations were performed to confirm results of the transcriptomic and proteomic analyses. In conclusion, RegA was found to regulate genes that encode all the biological functions. This makes the two-component system RegB/RegA as a main regulatory system required for adaptation of B. suis to oxygen depletion, which can contribute to the constraint of bacterial growth, characteristic of chronic infection.