In epithelial cells, the intracellular pathogen Brucella abortus escapes from the endocytic pathway, exploits the autophagic machinery of the host cell and establishes a unique replication niche in the endoplasmic reticulum. The molecular mechanisms underlying these processes are still poorly understood. Recently, a B. abortus type IV-related secretion system encoded by the virB operon has been described as being involved in the intracellular trafficking of the bacteria. In this study, we have analysed the intracellular pathway of B. abortus virB10 mutant strains by confocal microscopy. We demonstrate that a functional virB operon is essential for the biogenesis of the Brucella-containing vacuole. Polar mutation preventing the transcription of virB10 and downstream sequences did not allow Brucella to bypass the endocytic pathway. Consequently, polar mutant-containing vacuoles fused with lysosomes in which bacteria underwent a degradation process. In contrast, virB10 non-polar mutants were capable of avoiding interactions with the endocytic pathway but, diverging to wild-type Brucella, were unable to reach the endoplasmic reticulum to establish their intracellular replication niche and seemed to be recycled to the cell surface. Based on the two particular phenotypes described in this work, a model of maturation of the Brucella-containing vacuole is proposed.

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis. A key determinant for intracellular pathogenic bacteria to induce infectious diseases is their ability to avoid recognition by the host immune system. Although most microorganisms internalized by host cells are efficiently cleared, Brucella behave as a Trojan horse causing a zoonosis called brucellosis that affects both humans and animals. Here we show that pathogenic Brucella are able to target host cell defense mechanisms by controlling the function of the sentinels of the immune system, the dendritic cells. In particular, the Brucella TIR-containing protein (Btp1) targets the Toll-like receptor 2 activation pathway, which is a major host response system involved in bacterial recognition. Btp1 is involved in the inhibition of dendritic cell maturation. The direct consequence is a control of inflammatory cytokine secretion and antigen presentation to T lymphocytes. These bacterial proteins are not specific for Brucella and have been identified in other pathogens and may be part of a general virulence mechanism used by several intracellular pathogens to induce disease.

Histidine kinases, used for environmental sensing by bacterial two-component systems, are involved in regulation of bacterial gene expression, chemotaxis, phototaxis, and virulence. Flavin-containing domains function as light-sensory modules in plant and algal phototropins and in fungal blue-light receptors. We have discovered that the prokaryotes Brucella melitensis , Brucella abortus , Erythrobacter litoralis , and Pseudomonas syringae contain light-activated histidine kinases that bind a flavin chromophore and undergo photochemistry indicative of cysteinyl-flavin adduct formation. Infection of macrophages by B. abortus was stimulated by light in the wild type but was limited in photochemically inactive and null mutants, indicating that the flavin-containing histidine kinase functions as a photoreceptor regulating B. abortus virulence.

As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus ( virB ) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment.

Despite their high DNA identity and a proposal to group classical Brucella species as biovars of Brucella melitensis , the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters, as well as by a marked host range (e.g., Brucella suis for swine, B. melitensis for sheep and goats, and Brucella abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucella species and to B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions, and insertions supports previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis . With the exception of a dozen genes, the genetic complements of both B. abortus strains are identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggests that these inactivations may play an important role in the establishment of host specificity and may have been a primary driver of speciation in the genus Brucella . Despite being nonmotile, the brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures and may contribute to differences in host specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g., glycogen, biotin, NAD, and choline) are consistent with adaptation of brucellae to an intracellular life-style.

Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.