Identification of VceA and VceC, two members of the VjbR regulon that are translocated into macrophages by the Brucella type IV secretion system

Abstract

SummarySurvival and replication inside host cells by Brucella spp. requires a type IV secretion system (T4SS), encoded by the virB locus. However, the identity of the molecules secreted by the T4SS has remained elusive. We hypothesized that proteins translocated by the T4SS would be co-regulated with the virB operon. The LuxR family regulator VjbR, known to regulate virB, bound a fragment of the virB promoter containing an 18 bp palindromic motif (virB promoter box), showing that VjbR regulated the virB operon directly. To identify virB co-regulated genes, we searched the Brucella suis 1330 and B. abortus 2308 genomes for genes with an upstream virB promoter box. One hundred and forty-four promoters in the two genomes contained the virB promoter box, including those of fliC encoding flagellin and cgs encoding cyclic b-glucan synthetase. Thirteen of these proteins were tested for VirB-dependent translocation into macrophages using a b-lactamase reporter assay. This analysis resulted in the identification of the proteins encoded by BAB1_1652 (VceA) and BR1038/ BAB1_1058 (VceC) as novel protein substrates of the Brucella T4SS. VceC could also be translocated by the Legionella pneumophila Dot/Icm T4SS into host cells. Our results suggest that VjbR co-ordinates expression of the T4SS and at least two of its secreted substrates.

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

“…To date, 15 T4SS substrates have been identified (4 -8, 28), but we do not know the molecular mechanism that mediates their secretion and/or translocation. Interestingly, some of these effectors, like SepA, Bpe123, Bpe275, Bpe043, VceC, BspC, BspE, and BspB, either have a predicted periplasmic signal peptide or a transmembrane domain (5)(6)(7)(8), which highlights the complexity of the secretion process.…”

“…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.…”

“…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).…”

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

“…Until now, little has been known about the nature and the molecular functions of Brucella effectors. 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).…”