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, it is commonly accepted that intracellular brucellae avoid fusion of their vacuole, the Brucella-containing vacuole (BCV), with lysosomes via segregation from the degradative endocytic pathway. BCVs then interact with subcompartments of the endoplasmic reticulum, known as ER exit sites (ERES) and eventually fuse with the core ER to generate an ER-derived organelle permissive for replication. The VirB type IV secretion system, a major determinant of Brucella virulence that is induced inside the intermediate BCV, is required for the conversion of the BCV into a replicative organelle, likely through the translocation of effector molecules into the macrophage that modulate host functions. Yet, none of these effectors have been identified, impairing advances in the understanding of Brucella molecular pathogenesis. ? ? 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, using confocal live cell microscopy methods that have not yet been applied to Brucella. Due to the sensitivity of these techniques, we found that intermediate BCVs significantly interact with the late endosomal/lysosomal compartment, including lysosomes, as judged by the recruitment on BCV of the small GTPase Rab7, its effector RILP, and BCV accessibility to fluid phase markers delivered from lysosomes. Moreover, such interactions are required for further trafficking to the ER and bacterial replication, possibly by providing an inducing environment for expression of the VirB apparatus.? ? To identify VirB effector molecules and further characterize the molecular mechanisms of Brucella intracellular survival, we have hypothesized that genes encoding VirB effectors are co-regulated with the virB operon and could be identified based on their expression profiles inside macrophages. Because expression of the virB operon depends upon the luxR-family transcriptional regulator VjbR, we have hypothesized that expression of genes encoding VirB substrates is controlled by the same regulatory network. We have thus generated a vjbR deletion mutant in B. abortus and its chromosomal revertant in order to compare their global expression profiles with that of the wild type strain using comparative microarray analysis in VirB-inducing conditions. While we are optimizing our procedures to isolate quality bacterial RNA from infected murine macrophages in sufficient amounts to obtain robust microarray data, we are also performing such a comparative analysis using in vitro conditions that can mimic intravacuolar VirB-inducing cues and are not limiting in terms of bacterial RNA yields.? Additionally, we have been using bioinformatics tools in collaboration with the RTB/RTS Genomics Unit at RML to select genes that encode candidate effectors, based on their unicity to Brucella and the presence of motifs related to type IV secreted proteins in homologous VirB systems, or on the presence of eukaryotic-like motifs consistent with expected functions for these effectors. Fourteen candidates have been identified in the Brucella genome and are being tested for translocation into mammalian cells using an assay developed by the group of Renee Tsolis at the University of California at Davis.
| 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 |
