Animals; Bacterial Proteins/genetics/metabolism; Brucella abortus/pathogenicity; Cell Line; Epithelial Cells/microbiology; Gene Deletion; Host-Pathogen Interactions; Humans; Macrophages/microbiology; Mice; Phagosomes/metabolism/microbiology; Protein Binding; Protein Interaction Mapping; Two-Hybrid System Techniques; Virulence; Virulence Factors/genetics/metabolism; rab2 GTP-Binding Protein/metabolism
[en] Bacteria of the Brucella genus are facultative intracellular class III pathogens. These bacteria are able to control the intracellular trafficking of their vacuole, presumably by the use of yet unknown translocated effectors. To identify such effectors, we used a high-throughput yeast two-hybrid screen to identify interactions between putative human phagosomal proteins and predicted Brucella spp. proteins. We identified a specific interaction between the human small GTPase Rab2 and a Brucella spp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-beta-lactamase-RicA fusion was translocated from Brucella abortus to RAW264.7 macrophages during infection. This translocation was not detectable in a strain deleted for the virB operon, coding for the type IV secretion system. However, RicA secretion in a bacteriological culture was still observed in a DeltavirB mutant. In HeLa cells, a DeltaricA mutant recruits less GTP-locked myc-Rab2 on its Brucella-containing vacuoles, compared with the wild-type strain. We observed altered kinetics of intracellular trafficking and faster proliferation of the B. abortusDeltaricA mutant in HeLa cells, compared with the wild-type control. Altogether, the data reported here suggest RicA as the first reported effector with a proposed function for B. abortus.