Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages.Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional (5,14,17,28) and nonprofessional (9, 25, 26) phagocytic cells. Usually, phagosomes mature gradually into phagolysosomes which are capable of degrading proteins by lysosomal enzymes. In the case of

Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. We have previously shown that the maturation inhibition of the Brucella-containing phagosome appears to be restricted at the phagosomal membrane, but the precise molecular mechanisms and factors involved in this inhibition have yet to be identified. Interestingly, recent studies have revealed that caveolae or lipid rafts are implicated in the entry of some microorganisms into host cells and mediate an endocytic pathway avoiding fusion with lysosomes. In this study, we investigated the role of cholesterol and the ganglioside GM 1 , two components of lipid rafts, in entry and short-term survival of Brucella suis in murine macrophages, by using cholesterol-sequestering (filipin and ␤-methyl cyclodextrin) and GM 1 -binding (cholera toxin B) molecules. Our results suggest that lipid rafts may provide a portal for entry of Brucella into murine macrophages under nonopsonic conditions, thus allowing phagosome-lysosome fusion inhibition, and provide further evidence to support the idea that the phagosome maturation inhibition is restricted at the phagosomal membrane.Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional (4,14,16,26) and nonprofessional (8, 23, 24) phagocytic cells. In the classic endocytic pathway, phagosomes interact with lysosomes, allowing degradation by lysosomal enzymes. In the case of Brucella, inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival of the bacteria in both types of cells (1,10,14,18,23,24).The ability to inhibit phagosome maturation into a phagolysosome is shared by several bacteria and parasites, but the molecular mechanisms and the responsible microbial factors are poorly understood. This maturation inhibition has been associated with proteins secreted in the macrophage cytosol; for example, Salmonella SpiC protein is exported into the host cell cytosol and inhibits cellular trafficking (33). For Legionella pneumophila, the dot/icm gene products are required to avoid normal trafficking of the L. pneumophila phagosome (28, 35). For some other parasites, inhibition is associated with the presence of particular surface molecules on the organism's membrane or on the phagosomal membrane. Hence, for Leishmania, maturation inhibition requires lipophosphoglycan expression at the parasite surface (7, 29), and in mycobacteria, the TACO host protein present on the phagosomal membrane in a cholesterol-dependent manner prevents lysosomal delivery (9). It has previously been shown that the maturation inhibition of the Brucella-containing phagosome app...

Human Vγ9Vδ2 T cells are considered to play an important role in brucellosis, as this population is dramatically increased in peripheral blood of patients during the acute phase of the infection. This T lymphocyte population has been largely demonstrated to be activated by small m.w. nonpeptidic molecules from natural or synthetic origin. We recently identified a nonpeptidic fraction of Brucella suis that specifically activates human Vγ9Vδ2 T cells. Using a two-separate-chambers system, we showed that Brucella fraction, as well as isopentenyl pyrophosphate-activated Vγ9Vδ2 T cells, impaired the multiplication of B. suis in differentiated THP-1 cells through TNF-α and IFN-γ release. In the present study, using circulating Vγ9Vδ2 T cells and autologous monocytes infected with B. suis, we provide evidence that 1) intramonocytic multiplication of B. suis is impaired by supernatants of activated Vγ9Vδ2 T cells in part via TNF-α and IFN-γ, this impairment occurring without host cell lysis; 2) unstimulated Vγ9Vδ2 T cells can impair intracellular bacterial multiplication after their activation by soluble factors released by infected monocytes; and 3) activated Vγ9Vδ2 T cells lyse Brucella-infected monocytes in a contact-dependent manner. Taken together, these results provide evidence that Vγ9Vδ2 T cells, in addition to being directly activated by soluble nonpeptidic molecules, can be stimulated to become highly cytotoxic in the specific presence of infected monocytes; moreover, they suggest how Vγ9Vδ2 T cells could be triggered and respond as antibacterial effector cells in the early stages of Brucella infection.

Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic (7,15,18,27) and nonprofessional phagocytic (10, 24, 25) cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. Hence, several reports have described a decrease in the fusion of Brucella-containing phagosomes with lysosomes within macrophages (2,12,15,21). 25) also recently reported that virulent Brucella abortus avoids lysosome fusion in HeLa cells and replicates in endoplasmic reticulum-like structures.It has long been known that several bacteria and parasites can inhibit maturation of their phagosomes into phagolysosomes to enable survival and replication within host cells, but the responsible microbial factors have only been identified in a few cases. This maturation inhibition was found to be associated with proteins secreted in the macrophage cytosol; e.g., Salmonella SpiC protein is exported in the host cell cytosol and inhibits cellular trafficking (30). For other parasites, inhibition is associated with the presence of particular surface molecules on the microorganism membrane or on the phagosomal membrane. Hence in Leishmania, maturation inhibition requires lipophosphoglycan (LPG) expression at the parasite surface...