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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.

Scientific reports 7, 44420 (2017). doi:10.1038/srep44420, Published by Nature Publishing Group, London

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

International audience

Virulence 10(1), 868-878 (2019). doi:10.1080/21505594.2019.1682762

Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

International audience; In murine infections, Brucella microti exhibits an atypical and highly pathogenic behavior resulting in a mortality of 82%. In this study, the possible involvement of the virB type IV secretion system, a key virulence factor of Brucella sp., in this lethal phenotype was investigated. As previously described for B. suis, expression of the virB operon of B. microti was induced in acid minimal medium, partially mimicking intracellular environment. Early neutralization of cellular compartments abolished intracellular replication of B. microti, showing that acidity of the Brucella-containing vacuole is an essential trigger. A ΔvirB mutant of B. microti exhibited strong attenuation in murine and human macrophages in vitro. Interestingly, infection with this mutant was not lethal in Balb/c mice and lacked the typical intrasplenic peak at 3 days post-infection, hence demonstrating that lethality of B. microti in murine infection absolutely requires a functional virB operon.

The lymphoma of the mucosa-associated lymphoid tissue (MALT) of the stomach has been linked to Helicobacter pylori infection, but the mechanisms involved in B-cell proliferation remain elusive. In a search for putative H. pylori-specific monoclonal immunoglobulin production, an H. pylori strain was isolated from 10 patients with MALT lymphoma and used to detect the specific serum antibody response to the homologous strain by immunoblotting. Moreover, the antigenicity of the different strains was compared by using each of the 10 sera. We found that the different strains induced highly variable patterns of systemic immunoglobulin G antibody response, although several bacterial antigens, such as the 60-kDa urease B, were often recognized by the different sera. The cagA marker was detected in the strains by PCR with specific primers and by dot blot analysis, and the CagA protein was found in the sera of 4 of the 10 patients by immunoblotting. In conclusion, MALT lymphoma patients, like other patients with H. pylori gastritis, exhibit a polymorphic systemic antibody response, despite an apparently similar antigenic profile. The CagA marker of pathogenicity is not associated with this disease.

When the standard procedure for determining antibiotic susceptibility of bacteria is used, the results are delayed, especially for bacteria that grow slowly, such as Helicobacter pylori . Treatment for this bacterium may involve clarithromycin, a compound for which resistance has been associated with point mutations on the 23S rRNA gene. This resistance is currently found in organisms isolated from 0 to 15% of patients and jeopardizes the success of the treatment. We have designed a test involving amplification and colorimetric hybridization in the liquid phase to detect the mutation at the molecular level. First, four reference strains, including the wild type and three strains with the mutations A2143C, A2143G, and A2144G, were used to optimize the method. Amplification was carried out with primers previously published. The amplified products were added to probe-coated microtiter wells. A DNA enzyme immunoassay was used to detect the hybrids. The optimal conditions of the hybridization were defined for each probe. Nineteen H. pylori strains resistant to clarithromycin and 22 susceptible according to phenotypic data were submitted to restriction with Bsa I and Bbs I, and part of the 23S rRNA gene was sequenced in order to determine the mutation involved for the resistant strains. The new assay showed a complete correlation with the reference methods, except for one strain. Cross-hybridizations as well as application of the reaction to other bacteria did not lead to optical densities higher than the cutoff values chosen with the receiving operating characteristic curve. This method can be easily standardized and gives a result within a day. Its application directly to the biopsy specimens or infected gastric juice is planned in the future.

Macrolides and nitroimidazoles are among the antibiotics currently used for Helicobacter pylori eradication for which an acquired resistance may occur. Macrolides bind tightly to H. pylori ribosomes and inhibit protein synthesis. Resistance has been found associated with a lack of binding and to a point mutation in position 2058 or 2059 of the 23S ribosomal RNA genes (domain V). This resistance is easy to detect and is clinically relevant. There is a decrease of up to 40% in the success rate of triple therapies including clarithromycin when there is a resistance of H. pylori to this compound. Nitroimidazoles must be reduced to be active on the bacterial DNA. Resistance is associated with a lack of reduction of the NO2 group. But this reduction can be influenced by the redox potential of the environment. The genetic background is not yet known. There are discrepancies between the different methods used to test susceptibility to nitroimidazole; the best method which is now in the process of being standardized, is the agar dilution method. The clinical relevance of this resistance has been questioned. However, despite all the problems highlighted, there is still a difference between strains categorized as susceptible or resistant with regard to the eradication rate of H. pylori. This difference is greater when nitroimidazole is associated with amoxicillin rather than with clarithromycin.