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National audience; Objectifs : L’adaptation à l’hypoxie conditionne la pathogénicité de Brucella, bactérie aérobie stricte, qui doit faire face au manque d’O2 chez l’hôte. Notamment au stade chronique, Brucella peut persister dans des granulomes hypoxiques. Brucella suis dispose de nombreux systèmes spécifiques de l’hypoxie : deux oxydases terminales de haute affinité pour O2, le régulateur transcriptionnel FnrN, une voie de dénitrification complète et le système à deux composants RegB/A, senseur de l’état redox. L’étude fonctionnelle de ces systèmes a été développée pour analyser le rôle du régulateur transcriptionnel RegA dans leur contrôle et dans la persistance.Matériels et Méthodes : Le phénotype des mutants correspondants a été analysé in vitro sous différentes conditions d’hypoxie, in cellulo par infection de macrophages et in vivo, chez la souris. Un modèle de persistance in vitro a été mis au point pour l’étude comparative des transcriptomes et protéomes des souches sauvage et mutante ΔregA. Résultats principaux : Les deux systèmes respiratoires, respiration oxydative et dénitrification, sont nécessaires à la virulence ou à la persistance de B. suis in vivo. RegB/A joue un rôle central dans la régulation coordonnée de ces deux systèmes in vitro et RegA est essentiel pour l’infection chronique in vivo. Les analyses transcriptomiques comparatives dans le modèle de persistance in vitro ont montré que RegA agit sur 12% des gènes de B. suis, représentant toutes les classes fonctionnelles. Les protéines comme les gènes impliqués dans la production d’énergie comprennent le plus grand nombre de facteurs réprimés par RegA, dont aceA codant pour l'IsoCitrate Lyase (ICL) subit la plus forte répression. De plus, in vivo, la multiplication et la persistance en phase chronique de B. suis sont strictement dépendantes de l’activité ICL.Conclusions : Chez B. suis, RegA est un régulateur global contrôlant non seulement les systèmes respiratoires spécifiques de l’hypoxie mais également de nombreuses voies métaboliques. Sa fonction centrale contribue à l’installation de l’état de persistance non-proliférative, typique de l’infection chronique, en adaptant le métabolisme aux besoins alors réduits en énergie. L’activation de systèmes à deux composants contrôlant les régulons impliqués dans la persistance est probablement largement répandue parmi les bactéries aérobies pathogènes.

International audience; Oxygen deficiency is one important parameter during human chronic infection by Brucella, which can reside in hypoxic immune structures and become resistant to the clinically used antibiotics. Our studies demonstrated the high metabolic flexibility of Brucella suis with respect to oxygen deprivation. We evidenced the central role of the two-component system RegBA in the coordinated control of oxidative respiration and denitrification, and its requirement for B. suis persistence in mice. RegA was essential for optimal long-lasting persistence in our original in vitro model, characterized by progressive oxygen deprivation. With this model, global transcriptome and proteome analyses comparing the wild-type and regA B. suis strains showed that genes or proteins involved in secretion (type IV), envelope biogenesis and in division were down-regulated by RegA. Importantly, the greatest number of the RegA-repressed genes and proteins, including aceA encoding the functional IsoCitrate Lyase (ICL), were involved in energy production. Moreover, ICL is an essential determinant of pathogenesis and long-term interactions with the host, as demonstrated by the strict dependence of B. suis on ICL activity for multiplication and persistence in vivo. RegA-dependent regulation of genes and proteins belonging to all functional groups (potentially 12% of the B. suis genes) makes the two-component system RegBA a main regulatory system required for adaptation of B. suis to oxygen depletion. This function may contribute to the establishment of a non-replicative state, typical of chronic infection.

Frontiers in Cellular and Infection Microbiology 7, 186 (2017). doi:10.3389/fcimb.2017.00186, Published by Frontiers Media, Lausanne

International audience; 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.

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