Identification of a Brucella spp. secreted effector specifically interacting with human small GTPase Rab2

Bacteria of the Brucellagenus 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 screento identify interactions between putative human phagosomalproteins and predicted Brucellaspp. proteins. We identified a specific interaction between the human small GTPaseRab2 and a Brucellaspp. protein named RicA. This interaction was confirmed by GST-pull-down with the GDP-bound form of Rab2. A TEM-β-lactamase-RicA fusion was translocated from Brucella abortusto 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 ΔvirB mutant. In HeLa cells, a ΔricA 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. abortusΔricA 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.