In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

Abstract

SUMMARY Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.

VirJ Is a Brucella Virulence Factor Involved in the Secretion of Type IV Secreted Substrates

“…This is the mechanism by which they mediate secretion. An intriguing observation in several bacteria with T4SSs is that they are able to secrete and/or translocate substrates that are present in the cytoplasm, the periplasmic space, or even inserted in the inner membrane (6,7,11,(15)(16)(17)26). These reports raise issues regarding the initial models that proposed that the T4SSs engage their substrates in the cytoplasm and should encourage the search for alternative models.…”

“…We showed that this new effector protein is secreted in a VirB-dependent manner, that its inactivation affects the intracellular trafficking particularly during the initial stages, and that its secretion involves a periplasmic intermediate (6). Although in Brucella this was the first report identifying a VirB substrate with a twostep secretion process involving a periplasmic intermediate, it was reported that other effectors have a predicted periplasmic signal peptide or putative transmembrane domains (5,7,8). We report here the identification in Brucella of a homologue of the A. tumefaciens virJ gene and characterized this gene genetically and biochemically.…”

“…Construction of the Ba bpe123 and Ba ⌬virJ/bpe123 StrainsTo construct the Ba bpe123 and Ba ⌬virJ/bpe123 strains expressing Bpe123-3ϫFLAG, the plasmid pBPE123-FLAG (7) was introduced in the Ba wild-type and Ba ⌬virJ mutant strains by biparental mating. The expression of Bpe123-3ϫFLAG was confirmed by Western blotting.…”

“…Brucella is an intracellular pathogen with the capacity to avoid the bactericidal effects of its target cells, such as macrophages (one of the primary cell targets). To achieve this, the bacterium codes for a T4SS 5 that secretes and translocates effector proteins to the host cell that modulate the cellular response, avoiding the phagocytic process and favoring the infectious process (3)(4)(5)(6)(7)(8)(9)(10). In Brucella, this secretion system is named virB because of its homology to the Agrobacterium tumefaciens conjugation-like system that translocates the T-DNA (transferred DNA) into the plant cell (11).…”

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).…”