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

Abstract

SummaryBacteria 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-b-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 DvirB mutant. In HeLa cells, a DricA 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. abortus DricA 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.

A Brucella abortus cstA mutant is defective for association with endoplasmic reticulum exit sites and displays altered trafficking in HeLa cells

“…The secretion test and sample preparation for Western blotting were performed as described previously (de Barsy et al, 2011).…”

“…3c). Since strains affected for intracellular trafficking are not necessarily attenuated (de Barsy et al, 2011), we tested the co-localization of the DcstA strain with some typical markers of intracellular trafficking in HeLa cells. We first tested the ability of the DcstA strain to interact with ERES by localization of Sec23 and B. abortus during HeLa cell infection.…”

“…Several proteins were identified as T4SS substrates, but their functions remain unknown (de Jong et al, 2008;Marchesini et al, 2011). Recently, the RicA Brucella effector involved in Rab2 recruitment on the BCVs was identified using a yeast two-hybrid (Y2H) screen (de Barsy et al, 2011). Using a Y2H screen between a set of 15 human ERES-associated proteins and proteins encoded by the Brucella ORFeome , we identified CstA, a secreted Brucella protein that specifically interacts with human Sec24A.…”

Bartonella and Brucella--Weapons and Strategies for Stealth Attack

“…Examples are (1) VceA and VceC, which belong to the VjbR regulon, consequently having their expression coregulated with the VirB T4SS (de Jong et al 2008); (2) BPE123, whose translocation additionally depends on a Sec-secretion signal at the amino terminus; and (3) BPE005, BPE275, and BPE043 (Table 1) (Marchesini et al 2011). Other putative effectors with indefinite positively charged amino acid carboxy-terminal sequences are (1) RicA, which is the sole effector having an assigned host partner, the guanosine di-phosphate (GDP)-bound form of the small GTPase Rab2 (de Barsy et al 2011); (2) Btp1 (TcpB in B. melitensis), which regulates innate immune responses via its Toll/interleukin-1 receptor (TIR) domain (Cirl et al 2008;Salcedo et al 2008) and possibly modulates microtubule dynamics (Radhakrishnan et al 2011); (3) PrpA, a proline-racemase family member, which regulates immune responses (Spera et al 2006); (4) CstA (conserved Sec24A-targeted protein A), which controls Brucella intracellular trafficking (de Barsy et al 2012); and (5) BvfA, which is required for Brucella intracellular replication (Lavigne et al 2005).…”

Protein-protein Interactions: Network Analysis and Applications in Drug Discovery

“…A Brucella spp. protein named RicA was picked up as a specific interactor of the human small GTPase Rab2, which was previously shown to be essential for intracellular replication of B. abortus [157,158].…”