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

Development of vaccine candidates against Brucella melitensis and Acinetobacter baumannii infections in a mouse model

Facultative intracellular bacteria of the Brucella genus cause brucellosis, a zoonotic infectionwith a significant socioeconomic impact in southern countries. The recommended liveattenuated vaccines (LAVs) against brucellosis, Rev.1 and S19, provide satisfactory protectionfor livestock but can induce abortions and are virulent for humans. This situation causes seriouseconomic losses and human infections. These first-generation LAVs were generated byempirical methods such as random attenuation by successive passages. The transposonsequencing (Tn-seq) approach offers a new avenue for the rational development of safer LAVs.Indeed, the Tn-seq approach makes it possible to predict the genes required by the bacteria togrow in different conditions such as culture medium, cell infection (in vitro) and mice infection(in vivo). These genes can then be deleted to weaken the strain and try to select new LAVcandidates. Our results demonstrate that Brucella melitensis faces different selection pressuresdepending on the infected organ. Based on our Tn-seq data, we selected genes whose deletiongenerates strains capable of multiplying temporarily in the lungs and the spleen and inducingprotective immunity but without establishing themselves permanently in the spleen, the mainreservoir organ. We tested the persistence and the induced immune protective memory of a plsCgene deletion mutant, involved in the biosynthesis of membrane phospholipids. We observedthat this mutant induces similar protection but persists less in the spleen than the referenceRev.1 vaccine, which suggests that it could be safer.Acinetobacter baumannii is a bacterium responsible for serious nosocomial infections,including pneumonia, mainly affecting immunocompromised individuals. The outbreak of drugmulti-resistant A. baumannii strains to antibiotics makes the development of a vaccine againstthis pathogen essential. In an intranasal infection model in mice, vaccination with theinactivated LAC-4 or AB5075 strain induces the development of immunity against a challengewith the live LAC-4 strain. We observed that intranasal vaccination with the inactivated LAC4 strain partially protects mice genetically deficient for key elements of the adaptive immuneresponse as well as mice treated with cyclophosphamide, an immunosuppressive drugfrequently used in human treatment. These results suggest that vaccination with an inactivatedA. baumannii strain could protect at-risk populations of immunocompromised patients.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

PdeA is required for the rod shape morphology of Brucella abortus

Cyclic-di-GMP plays crucial role in the cell cycle regulation of the α-Proteobacterium Caulobacter crescentus. Here we investigated its role in the α-Proteobacterium Brucella abortus, a zoonotic intracellular pathogen. Surprisingly, deletion of all predicted cyclic-di-GMP synthesizing or degrading enzymes did not drastically impair the growth of B. abortus, nor its ability to grow inside cell lines. As other Rhizobiales, B. abortus displays unipolar growth from the new cell pole generated by cell division. We found that the phosphodiesterase PdeA, the ortholog of the essential polar growth factor RgsP of the Rhizobiale Sinorhizobium meliloti, is required for rod shape integrity but is not essential for B. abortus growth. Indeed, the radius of the pole is increased by 31 ± 1.7% in a ΔpdeA mutant, generating a coccoid morphology. A mutation in the cyclic-di-GMP phosphodiesterase catalytic site of PdeA does not generate the coccoid morphology and the ΔpdeA mutant kept the ability to recruit markers of new and old poles. However, the presence of PdeA is required in an intra-nasal mouse model of infection. In conclusion, we propose that PdeA contributes to bacterial morphology and virulence in B. abortus, but it is not crucial for polarity and asymmetric growth.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

The Brucella effector BspL targets the ER-associated degradation (ERAD) pathway and delays bacterial egress from infected cells

Perturbation of the endoplasmic reticulum (ER), a central organelle of the cell, can have critical consequences for cellular homeostasis. An elaborate surveillance system known as ER quality control ensures that cells can respond and adapt to stress via the unfolded protein response (UPR) and that only correctly assembled proteins reach their destination. Interestingly, several bacterial pathogens hijack the ER to establish an infection. However, it remains poorly understood how bacterial pathogens exploit ER quality-control functions to complete their intracellular cycle. Brucella spp. replicate extensively within an ER-derived niche, which evolves into specialized vacuoles suited for exit from infected cells. Here we present Brucella-secreted protein L (BspL), a Brucella abortus effector that interacts with Herp, a central component of the ERassociated degradation (ERAD) machinery. We found that BspL enhances ERAD at the late stages of the infection. BspL targeting of Herp and ERAD allows tight control of the kinetics of autophagic Brucella-containing vacuole formation, delaying the last step of its intracellular cycle and cell-to-cell spread. This study highlights a mechanism by which a bacterial pathogen hijacks ERAD components for fine regulation of its intracellular trafficking.SCOPUS: ar.jinfo:eu-repo/semantics/publishe