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, none of these 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., Traffic 9 (5): 678694). In 2009, 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 induces the Unfolded Protein Response (UPR), which reorganizes BCVs into endosomal organelles. While autophagy was expected to drive these vacuole modifications, given that it is induced by ER stress, we found that BCV conversion is enhanced when autophagy is inhibited, either in Atg5-/- macrophages (in collaboration with Dr Skip Virgin, Washington University, Saint-Louis, MO) or in the cancer HeLa cell line treated with siRNA against Atg5 or MAP-LC3. These results suggest a novel membrane trafficking process between the ER and endosomes that does not involve classic autophagy machineries and that could be associated with bacterial egress from infected cells and reinfection. Additionally, we have been pursuing collaboration with Dr Rene Tsolis on the identification of substrates of the VirB Type IV secretion and on the function of the Brucella VirB effector protein VceC. Using ectopic expression of VceC in the cancer HeLa cell line, we have shown that this fusion induces the specific vacuolation of the ER, potentially through its interaction with the ER chaperone Grp78/Bip, whose inhibition induces ER stress. We have also completed this year a collaborative work with Dr Craig Roys lab at Yale University, examining functional differences between Brucella and Legionella pneumophila interactions with the ER in the context of specific recruitment of bacterial effectors to the ER-derived organelles generated by both pathogens (Ninio et al., PLoS Pathog, 5(1): e1000278).
