Bacteria of the genus Brucella are the etiologic agents of brucellosis, a worldwide zoonosis that is highly transmissible to humans. Due to its high infectivity through inhalation, brucellae are included in the CDC Category B list of Select Agents. Virulence of this pathogen mostly depends upon its ability to survive and replicate within macrophages of the infected host. Following phagocytosis, intracellular brucellae avoid fusion of their vacuole, the Brucella-containing vacuole (BCV), with lysosomes via segregation from the degradative endocytic pathway. BCVs then interact and fuse with the endoplasmic reticulum (ER) to generate an ER-derived organelle permissive for replication. We have recently shown that BCV interactions with the ER occur at specific subdomains of this compartment, the ER exit sites, the integrity and functionality of which are required for ER membrane accretion and biogenesis of the BCV. The VirB type IV secretion system, a major determinant of Brucella virulence, is required for the conversion of the BCV into a replicative organelle, likely through the translocation of effector molecules into the macrophage to modulate host functions. Yet, none of these effectors have been identified, impairing advances in the understanding of Brucella molecular pathogenesis. To identify VirB effector molecules and further understand the molecular mechanisms of Brucella intracellular survival, we have hypothesized that genes encoding VirB effectors are co-regulated with the virB operon. We have established the expression profile of virB genes during Brucella intracellular cycle by quantitative RT-PCR and have optimized procedures to isolate bacterial RNA from infected macrophages, in order to establish the intracellular transcriptome of Brucella and define sets of genes that are co-regulated with the virB operon. A complementary approach is based on the findings that expression of the virB operon depends upon the luxR-family transcriptional regulator vjbR. Assuming that expression of genes encoding VirB substrates is controlled by the same regulatory network, we have generated a vjbR deletion mutant to be used in comparison with the wild type B. abortus strain to define the vjbR regulon by differential DNA microarray analysis. Additionally, we have been using bioinformatics to select genes that encode candidate effectors, based on their unicity to Brucella and motifs present in type IV secreted proteins in homologous VirB systems, or on the presence of eukaryotic-like motifs consistent with membrane fusion properties. Several candidates have been identified and have been fused to the adenylate cyclase-encoding gene cyaA in B. abortus to assess VirB-dependent translocation of the resulting chimeric proteins in infected macrophages, via detection of CyaA-dependent cAMP production in the host cell. In parallel with these efforts, we are pursuing a cell-biology based characterization of the host functions involved in Brucella intracellular trafficking. The proposed early segregation of BCV from the endocytic pathway is inconsistent with the fact that BCV acidification is required for bacterial survival, possibly through the induction of the virB operon. Given this discrepancy, we have further investigated BCV interactions with endocytic compartments and found significant fusion of BCV with late endosomes, as judged by the recruitment of the small GTPase Rab7 and its effector RILP, but not with lysosomes. Interestingly, such interactions are important for further replication, indicating i) a larger role of the endocytic machineries in Brucella intracellular survival than previously thought; ii) that inhibition of fusion with lysosomes is downstream of the recruitment of late endocytic fusion regulatory proteins.
| Celli, Jean (2012) LRSAM1, an E3 Ubiquitin ligase with a sense for bacteria. Cell Host Microbe 12:735-6 |
| Celli, Jean (2008) Intracellular localization of Brucella abortus and Francisella tularensis in primary murine macrophages. Methods Mol Biol 431:133-45 |
| Starr, Tregei; Ng, Tony W; Wehrly, Tara D et al. (2008) Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment. Traffic 9:678-94 |
