Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice

Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B .melitensis .Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B .melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B .melitensis in RAW 264.7 macrophages in vitro .As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B .melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and β oxidation of fatty acids are no longer required in Interleukin (IL)-17RA -/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B .melitensis in vivo .info:eu-repo/semantics/publishe

Route of Infection Strongly Impacts the Host-Pathogen Relationship

Live attenuated vaccines play a key role in the control of many human and animal pathogens. Their rational development is usually helped by identification of the reservoir of infection, the lymphoid subpopulations associated with protective immunity as well as the virulence genes involved in pathogen persistence. Here, we compared the course of Brucella melitensis infection in C57BL/6 mice infected via intraperitoneal (i.p.), intranasal (i.n.) and intradermal (i.d.) route and demonstrated that the route of infection strongly impacts all of these parameters. Following i.p. and i.n. infection, most infected cells observed in the spleen or lung were F4/80+ myeloid cells. In striking contrast, infected Ly6G+ neutrophils and CD140a+ fibroblasts were also observed in the skin after i.d. infection. The virB operon encoding for the type IV secretion system is considered essential to deflecting vacuolar trafficking in phagocytic cells and allows Brucella to multiply and persist. Unexpectedly, the ΔvirB Brucella strain, which does not persist in the lung after i.n. infection, persists longer in skin tissues than the wild strain after i.d. infection. While the CD4+ T cell-mediated Th1 response is indispensable to controlling the Brucella challenge in the i.p. model, it is dispensable for the control of Brucella in the i.d. and i.n. models. Similarly, B cells are indispensable in the i.p. and i.d. models but dispensable in the i.n. model. γδ+ T cells appear able to compensate for the absence of αβ+ T cells in the i.d. model but not in the other models. Taken together, our results demonstrate the crucial importance of the route of infection for the host pathogen relationship.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Inflammatory Properties and Adjuvant Potential of Synthetic Glycolipids Homologous to Mycolate Esters of the Cell Wall of Mycobacterium tuberculosis

&lt;p&gt;The cell wall of mycobacteria is characterised by glycolipids composed of different classes of mycolic acids (MAs; alpha-, keto-, and methoxy-) and sugars (trehalose, glucose, and arabinose). Studies using mutant Mtb strains have shown that the structure of MAs influences the inflammatory potential of these glycolipids. As mutant Mtb strains possess a complex mixture of glycolipids, we analysed the inflammatory potential of single classes of mycolate esters of the Mtb cell wall using 38 different synthetic analogues. Our results show that synthetic trehalose dimycolate (TDM) and trehalose, glucose, and arabinose monomycolates (TMM, GMM, and AraMM) activate bone marrow-derived dendritic cells in terms of the production of pro-inflammatory cytokines (IL-6 and TNF-&amp;alpha;) and reactive oxygen species, upregulation of costimulatory molecules, and activation of NLRP3 inflammasome by a mechanism dependent on Mincle. These findings demonstrate that Mincle receptor can also recognise pentose esters and seem to contradict the hypothesis that production of GMM is an escape mechanism used by pathogenic mycobacteria to avoid recognition by the innate immune system. Finally, our experiments indicate that TMM and GMM, as well as TDM, can promote Th1 and Th17 responses in mice in an OVA immunisation model, and that further analysis of their potential as novel adjuvants for subunit vaccines is warranted.&lt;/p&gt;</p

Identification des gènes bactériens indispensables lors d’une infection pulmonaire par Brucella dans le modèle expérimental murin

La brucellose est infection causée par les bactéries du genre Brucella. Cette maladie est répandueà travers le monde entier chez les mammifères et est transmissible aux humains. Causant notammentstérilité, avortement et destruction des articulations, la brucellose représente un risque sanitaire majeur.La chronicité et la récurrence de cette infection provoquent une morbidité importante chez l'hommemalgré des traitements antibiotiques longs et coûteux. Actuellement, les vaccins disponibles ne sont pasconsidérés comme sûrs et efficaces. Le confinement de la maladie repose en partie sur l'identification etl’élimination des troupeaux infectés. La brucellose représente toujours d'énormes pertes économiquespour les pays où la maladie est endémique. Le développement rationnel d'un vaccin atténué efficace etsûr contre les infections à Brucella nécessite l'identification des gènes de virulence indispensables à laréplication in vivo de la bactérie.Dans un modèle d'infection intranasale bien caractérisé chez la souris imitant l'infection naturellepar voie aérienne, nous avons décrit la dynamique de l'infection. En utilisant un marqueur fluorescent,nous sommes en mesure de surveiller la multiplication bactérienne in situ et de déterminer les différentesphases de l'infection. Lors d'une infection intranasale, les macrophages alvéolaires (MA) sont le principaltype cellulaire infecté mais seule une petite proportion de la MA infectée (5 à 15%) est permissive àl'infection. Les bactéries entrant dans la réplication au cours des premières 24 heures sont massivementéliminées, mais cette importante pression sélective peut être partiellement levée par des déficitsimmunitaires génétiques par exemple pour la signalisation de l’IL-17 (réponse immunitaire de type TH17)ou même par une altération de la réponse immunitaire en induisant un phénotype asthmatique (réponseimmunitaire de type TH2).Une identification approfondie de tous les gènes essentiels nécessaires à la croissance sur desmilieux riches ou des gènes conditionnellement nécessaires à la survie lors d'une infection in vitro(macrophages RAW murins) ou in vivo (souris) a été effectuée à différents moments clés précoces du cycleinfectieux en utilisant la technique du séquençage des transposons (Tn-Seq). Sur les 3140 gènes de B.melitensis, 643 sont requis pour la croissance extracellulaire sur des milieux riches. 179 gènessupplémentaires sont indispensables à la survie dans les poumons de la souris jusqu'à 5 jours aprèsl'infection. Seule la moitié de ces gènes peuvent être identifiés à l'aide du modèle in vitro standard,illustrant la limitation d'une telle approche in vitro pour identifier les exigences d'adaptation àl'environnement hôte. L'application de l'analyse en cluster montre que la plupart de ces gènes identifiéspeuvent être recadrés en voies complètes ou impliqués dans des fonctions liées. La synthèse deslipopolysaccharides, la synthèse de certains acides aminés, la B oxydation des acides gras et la cytochromeC oxydase seraient particulièrement importantes face à l'environnement hôte. Nous avons maintenantune idée plus claire des exigences minimales pour que la bactérie infecte avec succès son hôte. Enappliquant cette approche en cas d’immunodéficience pour la signalisation de l’IL-17 ou en conditionasthmatique, nous savons maintenant que l'essentialité de certains gènes peut être levée à savoir lasynthèse du core et de la chaîne O du lipopolysaccharide et la B oxydation des acides gras respectivement.La délétion génétique de certains gènes sélectionnés (10) candidats valide les résultats de nos analysesTn-Seq. Ces analyses comparatives ont le potentiel d'identifier des souches de mutants atténués quipourraient déclencher une immunité protectrice sans la capacité de se propager ou de devenir chroniqueou d'être entièrement virulente même chez des animaux avec une immunité compromise.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Identification of immune effectors essential to the control of primary and secondary intranasal infection with brucella melitensis in mice

The mucosal immune system represents the first line of defense against Brucella infection in nature. We used genetically deficient mice to identify the lymphocytes and signaling pathways implicated in the control of primary and secondary intranasal infection with B. melitensis. Our analysis of primary infection demonstrated that the effectors implicated differ at the early and late stages and are dependent on the organ. TCR-d, TAP1, and IL-17RA deficiency specifically affects early control of Brucella in the lungs, whereas MHC class II (MHCII) and IFN-γR deficiency impairs late control in the lungs, spleen, and liver. Interestingly, IL-12p352/2 mice display enhanced Brucella growth in the spleen but not in the lungs or liver. Secondary intranasal infections are efficiently contained in the lung. In contrast to an i.p. infectious model, in which IL-12p35, MHCII, and B cells are strictly required for the control of secondary infection, we observed that only TCR-b deficiency or simultaneous neutralization of IL-12p35-and IL-17A-dependent pathways impairs the memory protective response against a secondary intranasal infection. Protection is not affected by TCR-d, MHCII, TAP1, B cell, IL-17RA, or IL-12p35 deficiency, suggesting that CD4+ and CD8+ a/b+ T cells are sufficient to mount a protective immune response and that an IL-17A-mediated response can compensate for the partial deficiency of an IFN-γ-mediated response to control a Brucella challenge. These findings demonstrate that the nature of the protective memory response depends closely on the route of infection and highlights the role of IFN-γ-and IL-17RA-mediated responses in the control of mucosal infection by Brucella.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Investigation of inflammatory and allergic responses to common mold species: Results from in vitro experiments, from a mouse model of asthma, and from a group of asthmatic patients

Most studies on molds focus on Alternaria alternata and Aspergillus fumigatus. Here, we report on inflammatory and allergenic properties of more typical indoor species Aspergillus versicolor, P. chrysogenum, C. cladosporioïdes, and C. sphaerospermum that were compared to A. alternata and A. fumigatus. In a mouse model, after intranasal instillation, A. alternaria, A. versicolor, and C. sphaerospermum induced the early recruitment of neutrophils and the strong expression of inflammatory markers in the bronchoalveolar lavages fluids. A. fumigatus also induced the early accumulation of neutrophils but with lower levels of inflammatory markers. Chronic treatment induced variable response according to species: P. chrysogenum and A. fumigatus appeared strong pro-allergenic inducers compared to A. alternata and C. sphaerospermum while A. versicolor and C. cladosporioides induced a mixed pro-allergenic/pro-inflammatory response. In mold-sensitized asthmatics, mold-specific Immunoglobulin E (IgE) were detected with an in-house dot-blot assay. A. fumigatus and A. alternata were the most frequent sensitizers. Altogether, P. chrysogenum, P. brevicompactum, C. sphaerospermum, and C. cladosporïoides were the “major sensitizer” (defined as the strongest response against a single mold species) for almost 30% of the asthmatics. These results show that, not only A. alternata and A. fumigatus, but also indoor species have strong inflammatory and allergic properties and a harmful potency.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Trypanosoma infection favors Brucella elimination via IL-12/IFNγ-dependent pathways

This study develops an original co-infection model in mice using Brucella melitensis, the most frequent cause of human brucellosis, and Trypanosoma brucei, the agent of African trypanosomiasis. Although the immunosuppressive effects of T. brucei in natural hosts and mice models are well established, we observed that the injection of T. brucei in mice chronically infected with B. melitensis induces a drastic reduction in the number of B. melitensis in the spleen, the main reservoir of the infection. Similar results are obtained with Brucella abortus- and Brucella suis-infected mice and B. melitensis-infected mice co-infected with Trypanosoma cruzi, demonstrating that this phenomenon is not due to antigenic cross-reactivity. Comparison of co-infected wild-type and genetically deficient mice showed that Brucella elimination required functional IL-12p35/IFNγ signaling pathways and the presence of CD4 T cells. However, the impact of wild type and an attenuated mutant of T. brucei on B. melitensis were similar, suggesting that a chronic intense inflammatory reaction is not required to eliminate B. melitensis. Finally, we also tested the impact of T. brucei infection on the course of Mycobacterium tuberculosis infection. Although T. brucei strongly increases the frequency of IFNγCD4 T cells, it does not ameliorate the control of M. tuberculosis infection, suggesting that it is not controlled by the same effector mechanisms as Brucella. Thus, whereas T. brucei infections are commonly viewed as immunosuppressive and pathogenic, our data suggest that these parasites can specifically affect the immune control of Brucella infection, with benefits for the host