Innate immune recognition of flagellin limits systemic persistence of Brucella

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, since a flagellin-deficient mutant of B. melitensis was found to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunologic standoff between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology

Morphological analysis of the sheathed flagellum of Brucella melitensis

<p>Abstract</p> <p>Background</p> <p>It was recently shown that <it>B. melitensis </it>is flagellated. However, the flagellar structure remains poorly described.</p> <p>Findings</p> <p>We analyzed the structure of the polar sheathed flagellum of <it>B. melitensis </it>by TEM analysis and demonstrated that the Ryu staining is a good method to quickly visualize the flagellum by optical microscopy. The TEM analysis demonstrated that an extension of the outer membrane surrounds a filament ending by a club-like structure. The Δ<it>ftcR</it>, Δ<it>fliF</it>, Δ<it>flgE </it>and Δ<it>fliC </it>flagellar mutants still produce an empty sheath.</p> <p>Conclusions</p> <p>Our results demonstrate that the flagellum of <it>B. melitensis </it>has the characteristics of the sheathed flagella. Our results also suggest that the flagellar sheath production is not directly linked to the flagellar structure assembly and is not regulated by the FtcR master regulator.</p

Le flagelle de Brucella melitensis : caractérisation de la structure et de la régulation flagellaire

The bacterial flagellum is a rotative filamentous appendage which is anchored in the bacterial membranes and allows the bacterium to move. Besides its main locomotive function, the bacterial flagellum fulfills other functions such as adhesion and secretion. The filament, which comprises most of the flagellum structure, is formed by an assembly of proteins called flagellins. It is estimated that nearly 80% of the bacteria produce a flagellum. Brucella is a mammal pathogens bacterium that had long been designated as non-flagellated. However, recent discoveries have shown that all flagellar genes were present in its genome and were necessary to Brucella’s pathogenic ability. Several regulators that control the production of flagellar proteins have also been uncovered. However, no flagellum was viewed on Brucella until today. In this work, we showed for the first time through the optical and electron microscopy that B. melitensis produces transitionally a polar sheathed flagellum. On the other hand, we also investigated the regulation of flagellar system. Firstly, we studied the role of sigma factor RpoE1 on flagellar genes. The rpoE1 mutant overexpresses flagellar genes and produces a longer flagellum. Secondly, we showed that the production of flagellin was controlled by specific regulators named FlbT and FlaF. FlbT is a flagellin activator while FlaF is a flagellin inhibitor. In addition, we showed that the function of FlbT appears to be conserved among the Rizhobiales. We also showed that B. melitensis is more virulent in the absence of flagellin, which indicates a specific role of flagellin. The function of the flagellin of Brucella remains to be investigated. Finally, a preliminary in silico analysis of the genome of Brucella has enabled us to target other potential regulators of the flagellar system. The data gathered during this work have allowed us to draw a hierarchical regulatory cascade of flagellar genes of Brucella in 3 classes.Le flagelle bactérien est un appendice rotatoire filamenteux ancré dans les membranes bactériennes et permet à la bactérie de se mouvoir. Bien que le rôle locomoteur soit principal, d’autres rôles lui sont attribués comme l’adhérence et la sécrétion. Le filament compose la plus grande partie du flagelle. Celui-ci est formé par un assemblage de protéines appellées flagellines. On estime que près de 80% des bactéries produisent un flagelle. Brucella est une bactérie pathogène de mammifères qui a longtemps été désignée comme non-flagellée. Cependant, les récentes découvertes ont montré que tous les gènes flagellaires étaient présents dans son génome et qu’ils étaient nécessaires lors de l’infection. Plusieurs régulateurs qui contrôlent la production des protéines flagellaires ont également été mis au jour. Néanmoins, aucun flagelle n’avait encore été visualisé chez Brucella. Lors de ce travail, nous avons montré pour la première fois grâce à la microscopie optique et électronique que B. melitensis produit de manière transitoire un flagelle polaire de type gainé. D’autre part, nous avons aussi investigué la régulation du système flagellaire. Premièrement, nous avons étudié le rôle du facteur sigma RpoE1 sur les gènes flagellaires. Le mutant rpoE1 surexprime les gènes flagellaires et produit des flagelles plus longs. Deuxiemement, nous avons montré que la production de la flagelline était controlée par des régulateurs spécifiques nommés FlbT et FlaF. FlbT est un activateur de la production de la flagelline alors que FlaF est un inhibiteur. De plus, nous avons montré que la fonction de FlbT semble conservée parmi les Rizhobiales. Nous avons également montré que B. melitensis était plus virulente en absence de flagelline, ce qui montre un rôle spécifique de la flagelline qui reste à être investigué. Pour terminer, une analyse préliminaire in silico du gènome de Brucella nous a permis de cibler d’autres régulateurs potentiels du système flagellaire. Les données récoltées lors de ce travail nous ont permis de dessiner un schéma de régulation hiérarchique des gènes flagellaires de Brucella en 3 classes.(DOCSC03 ) -- FUNDP, 200

A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis.

Both a type IV secretion system and a flagellum have been described in Brucella melitensis. These two multimolecular surface appendages share several features. Their expression in bacteriological medium is growth curve dependent, both are induced intracellularly and are required for full virulence in a mouse model of infection. Here we report the identification of VjbR, a quorum sensing-related transcriptional regulator. A vjbR mutant has a downregulated expression of both virB operon and flagellar genes either during vegetative growth or during intracellular infection. In a cellular model, the vacuoles containing the vjbR mutant or a virB mutant are decorated with the same markers at similar times post infection. The vjbR mutant is also strongly attenuated in a mouse model of infection. As C(12)-homoserine lactone pheromone is known to be involved in virB repression, we postulated that VjbR is mediating this effect. In agreement with this hypothesis, we observed that, as virB operon, flagellar genes are controlled by the pheromone. All together these data support a model in which VjbR acts as a major regulator of virulence factors in Brucella