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 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 (R(X)7-8RXRXR within the last 25 amino-acids). Of ~ 3,200 open reading frames identified in the Brucella abortus genome, 8 candidate genes have been selected. We are expressing their fusion to the adenylate cyclase-encoding gene cyaA in B. abortus to assess VirB-dependent translocation of these bacterial proteins in infected macrophages via detection of CyaA-dependent cAMP production in the host cell. Genes encoding VirB substrates are likely co-regulated with the virB operon. We have established the expression profile of virB genes during Brucella intracellular cycle by quantitative RT-PCR and are now developing a microarray-based approach to define sets of genes that are co-regulated with the virB operon. Expression of the virB operon depends upon the luxR-family transcriptional regulator vjbR, and we hypothesized that expression of genes encoding VirB substrates is controlled by the same regulatory network. We are therefore generating a vjbR deletion mutant to be used in comparison with the wild type B. abortus strain to define the vjbR regulon by differential microarray analysis.
| 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 |
