Bacteria of the genus Brucella are the etiologic agents of brucellosis, a worldwide zoonosis 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 22 candidates, 5 have shown positive translocation in a variety of assays, demonstrating their translocation inot host cells during the infection 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.
