Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

Abstract

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis.

Pathogenesis and Immunobiology of Brucellosis

“…Candidate effectors were shown to target secretory pathway compartments when expressed ectopically and impaired host protein secretion. 31,39 Genetic studies have found a redundancy of function among effector candidates, consistent with the failure to identify effectors by using early genetic screens. Although several factors are now identified, the list is far from complete, and the mechanism by which replication is enhanced and lysosomal fusion is prevented remains undefined.…”

“…Although shown to be required to prevent trafficking to the lysosome, the mechanistic steps involved, including interacting partners, enzymatic reactions, protein modifications, and detailed intracellular trafficking, are only now being described after a series of experiments in which Brucella gene reporter fusions were found to be secreted in a T4SS-dependent manner. 23,25,39 Putative effector candidates were identified in silico on the basis of several criteria, including shared features with effectors expressed by other bacteria, eukaryotic motifs, GC content, and limited distribution across bacterial genera. Candidate effectors were shown to target secretory pathway compartments when expressed ectopically and impaired host protein secretion.…”

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).…”