Bacteria of the genus Brucella are the etiologic agents of brucellosis, the most widespread zoonosis in the world that is highly transmissible to humans. Due to its high infectivity through inhalation, brucellae are included in the CDC Category B list of Select Agents. Virulence of this pathogen mostly depends upon its ability to survive and replicate within macrophages of the infected host. Following phagocytosis, intracellular brucellae reside in a membrane-bound vacuole, the Brucella-containing vacuole (BCV), that progressively mature over several hours into a replicative organelle derived from the host endoplasmic reticulum (ER) where the bacteria proliferate. The VirB type IV secretion system, a major determinant of Brucella virulence that is induced intracellularly, is required for the conversion of the BCV into a replicative organelle, likely through the translocation of effector molecules into the macrophage that modulate host functions. Yet, very few effectors have been identified, impairing advances in the understanding of Brucella molecular pathogenesis. We have recently shown that BCVs mature along the endocytic pathway and fuse in a limited manner with terminal lysosomes. These events provide intravacuolar cues necessary to the expression of the VirB Type IV secretion system, and consequently, biogenesis of the replicative organelle (Starr et al., 2008 Traffic 9(5): 678). To identify Brucella proteins translocated by the VirB secretion machinery, we have used bioinformatics analyses and identified candidate genes based on conserved motifs of Type IV effectors and the presence of eukaryotic protein domains. Among 23 candidates, 11 have shown positive translocation in a variety of assays, demonstrating their translocation into host cells during the infection cycle. Most of these proteins are translocated into host cells in a VirB-dependent manner and several target the host secretory pathway and affect its functionality, consistent with Brucella subversion of this compartment during its intracellular cycle. In our efforts to better understand the Brucella infection cycle, we have further examined late events in the Brucella intracellular cycle in the context of bacterial egress following intracellular proliferation. We have found that Brucella proliferation in the ER is followed by bacterial translocation into endosomal organelles with ultrastructural features of autophagy. Interestingly, this process required the autophagy initiation proteins Beclin 1 and Ulk1 but was independent of the autophagy elongation proteins ATG5, ATG7, ATG4B ATG16- and LC3B in both primary macrophages and HeLa cells, demonstrating selective subversion of autophagy proteins by this bacterium. Moreover, formation of these autopahgic vacuoles was important for completion of the Brucella intracellular cycle and reinfection events. Altogether, these results demonstrate for the first time the subversion of the innate immune autophagic process for bacterial spread during infection (Starr et al., 2012 Cell Host Microbe, 11(1): 33-45). As mentioned previously, 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. In FY 2013, 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 -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.
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