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Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis , a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B . melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella , and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B . melitensis . The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B . melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates.

It is assumed that intracellular pathogenic bacteria have to cope with DNA alkylating stress within host cells. Here we use single-cell reporter systems to show that the pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophage infection. Genes encoding direct repair and base-excision repair pathways are required by B. abortus to face this stress in vitro and in a mouse infection model. Among these genes, ogt is found to be under the control of the conserved cell-cycle transcription factor GcrA. Our results highlight that the control of DNA repair in B. abortus displays distinct features that are not present in model organisms such as Escherichia coli . It is assumed that intracellular pathogenic bacteria must cope with DNA alkylating stress within host cells. Here, Poncin et al. show that the pathogen Brucella abortus does encounter alkylating stress within macrophages, and shed light into the pathways required for DNA repair in this organism.

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 .

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 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 operon encoding for the type IV secretion system is considered essential to deflecting vacuolar trafficking in phagocytic cells and allows to multiply and persist. Unexpectedly, the Δ 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 challenge in the i.p. model, it is dispensable for the control of 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.

Allergic asthma is a chronic Th2 inflammatory disease of the lower airways affecting a growing number of people worldwide. The impact of infections and microbiota composition on allergic asthma has been investigated frequently. Until now, however, there have been few attempts to investigate the impact of asthma on the control of infectious microorganisms and the underlying mechanisms. In this work, we characterize the consequences of allergic asthma on intranasal (i.n.) infection by bacteria in mice. We observed that i.n. sensitization with extracts of the house dust mite or the mold ( ) significantly increased the number of , and in the lungs of infected mice. Microscopic analysis showed dense aggregates of infected cells composed mainly of alveolar macrophages (CD11c F4/80 MHCII ) surrounded by neutrophils (Ly-6G ). Asthma-induced susceptibility appears to be dependent on CD4 T cells, the IL-4/STAT6 signaling pathway and IL-10, and is maintained in IL-12- and IFN-γR-deficient mice. The effects of the sensitization protocol were also tested on and pulmonary infections. Surprisingly, we observed that sensitization strongly increases the survival of infected mice by a T cell and STAT6 independent signaling pathway. In contrast, the course of infection is not affected in the lungs of sensitized mice. Our work demonstrates that the impact of the same allergic sensitization protocol can be neutral, negative, or positive with regard to the resistance of mice to bacterial infection, depending on the bacterial species.

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

Live attenuated vaccines (LAVs) whose virulence would be controlled at the tissue level could be a crucial tool to effectively fight intracellular bacterial pathogens, because they would optimize the induction of protective immune memory while avoiding the long-term persistence of vaccine strains in the host. Rational development of these new LAVs implies developing an exhaustive map of the bacterial virulence genes according to the host organs implicated. We report here the use of transposon sequencing to compare the bacterial genes involved in the multiplication of Brucella melitensis, a major causative agent of brucellosis, in the lungs and spleens of C57BL/6 infected mice. We found 257 and 135 genes predicted to be essential for B. melitensis multiplication in the spleen and lung, respectively, with 87 genes common to both organs. We selected genes whose deletion is predicted to produce moderate or severe attenuation in the spleen, the main known reservoir of Brucella, and compared deletion mutants for these genes for their ability to protect mice against challenge with a virulent strain of B. melitensis. The protective efficacy of a deletion mutant for the plsC gene, implicated in phospholipid biosynthesis, is similar to that of the reference Rev.1 vaccine but with a shorter persistence in the spleen. Our results demonstrate that B. melitensis faces different selective pressures depending on the organ and underscore the effectiveness of functional genome mapping for the design of new safer LAV candidates.