[Abstract unavailable

This review of Brucellaehost interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion systemdependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of next-generation sequences of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent targets for improved, safer brucellosis vaccines and therapeutics. It is noteworthy that long ago in his publication Epidemics, Hippocrates described brucellosis-type syndromes in humans living in the Mediterranean littoral. Many centuries later, British physician, David Bruce, and Greek physician, Themistokles Zammit, in 1886 would discover the causative agent, Micrococcus melitensis, of brucellosis and would identify milk products of goats as the source of infection for military troops on the island of Malta. Even after more than a century of extensive research, Brucella spp. are still serious animal pathogens that cause brucellosis, a zoonosis that results in substantial economic losses, human morbidity, and perpetuates poverty worldwide.1 These Gram-negative bacteria infect a diverse array of land and aquatic mammals,

Brucella species are facultative intracellular bacterial pathogens that cause brucellosis, a global zoonosis of profound importance. Although recent studies have demonstrated that Brucella spp. replicate within an intracellular compartment that contains endoplasmic reticulum (ER) resident proteins, the molecular mechanisms by which the pathogen secures this replicative niche remain obscure. Here, we address this issue by exploiting Drosophila S2 cells and RNA interference (RNAi) technology to develop a genetically tractable system that recapitulates critical aspects of mammalian cell infection. After validating this system by demonstrating a shared requirement for phosphoinositide 3-kinase (PI3K) activities in supporting Brucella infection in both host cell systems, we performed an RNAi screen of 240 genes, including 110 ER-associated genes, for molecules that mediate bacterial interactions with the ER. We uncovered 52 evolutionarily conserved host factors that, when depleted, inhibited or increased Brucella infection. Strikingly, 29 of these factors had not been previously suggested to support bacterial infection of host cells. The most intriguing of these was inositol-requiring enzyme 1 (IRE1), a transmembrane kinase that regulates the eukaryotic unfolded protein response (UPR). We employed IRE1α−/− murine embryonic fibroblasts (MEFs) to demonstrate a role for this protein in supporting Brucella infection of mammalian cells, and thereby, validated the utility of the Drosophila S2 cell system for uncovering novel Brucella host factors. Finally, we propose a model in which IRE1α, and other ER-associated genes uncovered in our screen, mediate Brucella replication by promoting autophagosome biogenesis.

Membrane tubules of uniform diameter (60-80 nm) and various lengths (up to several micrometers) emanate from elements of the Golgi stack and trans Golgi network (TGN). These organelle membrane tubules are thought to be involved in membrane trafficking and maintenance of Golgi͞TGN architecture. The number of these tubules, and their frequency of formation, can be greatly enhanced by the fungal metabolite brefeldin A (BFA), an inhibitor of Golgi͞TGN-associated coated vesicle formation. We show here that BFA stimulation of Golgi and TGN membrane tubulation, and the resultant retrograde transport of resident Golgi enzymes to the endoplasmic reticulum, was potently inhibited by a number of membrane-permeant antagonists of phospholipase A 2 (PLA 2 ; EC 3.1.1.4) activity. In addition, PLA 2 inhibitors on their own caused a reversible fragmentation of the Golgi complex into juxtanuclear, stacked cisternal elements. We conclude from these observations that tubulation of Golgi complex and TGN membranes requires a PLA 2 activity, and that this activity may participate not only in Golgi tubule-mediated retrograde trafficking to the endoplasmic reticulum, but also in the maintenance of Golgi complex architecture.