Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages - PubMed (original) (raw)

Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages

Françoise Porte et al. Infect Immun. 2003 Mar.

Abstract

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.

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Figures

FIG. 1.

FIG. 1.

Interaction of live or killed _B. suis_- and latex bead-containing phagosomes with lysosomes in murine macrophages. Lysosomes were labeled with TRD. Live and gentamicin (Genta)-killed B. suis expressed GFP; heat-killed B. suis were labeled with fluorescein. Bacterium and latex bead internalization was performed for 45 min. Cells were fixed at different time points after infection. (A) Confocal images of cells containing live or heat- or gentamicin-killed B. suis or latex beads obtained at 1 h postinfection. (B) Percentage of phagosome-lysosome fusion at different time points after infection. Fusion was evaluated by the colocalization of both markers, GFP or fluorescein and TRD. To determine the percentage of fusion, 100 to 200 bacteria were analyzed at each time point. Results were calculated from two independent experiments. Values are given as mean percentages of fusion ± standard deviations.

FIG. 2.

FIG. 2.

Growth of B. suis (wild-type strain) and B. suis mutants in murine macrophages. (A) Rough mutants: B. suis manB (manB mutant), B. suis 18E10 (wbdA mutant), and B. suis 20C2 (lpsA mutant). (B) VirB mutant: B. suis virB5 (virB5 mutant). Cells were infected, and at the indicated times postinfection (p.i.), the number of intracellular bacteria was determined as described in Materials and Methods. The data points are the means ± standard deviations of two wells. These data sets are representative of at least two independent experiments with similar results.

FIG. 3.

FIG. 3.

Interaction of Brucella rough strain-containing phagosomes with lysosomes in murine macrophages. Lysosomes were labeled with TRD. Bacteria expressed GFP. Cells were infected for 45 min and fixed at different time points after infection. (A) Confocal images of cells containing B. canis, B. suis manB (manB mutant), B. melitensis B3B2 (rfbE mutant), B. suis 18E10 (wbdA mutant), and B. suis 20C2 (lpsA mutant) obtained at 1 h postinfection. (B) Percentage of phagosome-lysosome fusion for each strain at different time points after infection. Fusion was evaluated by the colocalization of the both markers, GFP and TRD. To determine the percentage of fusion, 100 to 200 bacteria were analyzed for each strain at each time point. Values are given as mean percentages of fusion ± standard deviations.

FIG. 3.

FIG. 3.

Interaction of Brucella rough strain-containing phagosomes with lysosomes in murine macrophages. Lysosomes were labeled with TRD. Bacteria expressed GFP. Cells were infected for 45 min and fixed at different time points after infection. (A) Confocal images of cells containing B. canis, B. suis manB (manB mutant), B. melitensis B3B2 (rfbE mutant), B. suis 18E10 (wbdA mutant), and B. suis 20C2 (lpsA mutant) obtained at 1 h postinfection. (B) Percentage of phagosome-lysosome fusion for each strain at different time points after infection. Fusion was evaluated by the colocalization of the both markers, GFP and TRD. To determine the percentage of fusion, 100 to 200 bacteria were analyzed for each strain at each time point. Values are given as mean percentages of fusion ± standard deviations.

FIG. 4.

FIG. 4.

Interaction of smooth avirulent B. suis mutant-containing phagosomes with lysosomes in murine macrophages. Lysosomes were labeled with TRD, and bacteria expressed GFP. Cells were infected for 45 min and fixed at different time points after infection. (A) Confocal images of cells containing B. suis virB5 (virB5 mutant) obtained at 1 h postinfection. (B) Percentage of phagosome-lysosome fusion at different time points after infection. Fusion was evaluated by the colocalization of both markers, GFP and TRD. To determine the percentage of fusion, 100 to 200 bacteria were analyzed for each strain at each time point. Values are given as mean percentages of fusion ± standard deviations.

FIG. 5.

FIG. 5.

Effect of filipin, β-methyl cyclodextrin, and cholera toxin B subunit on short-term survival of nonopsonized and opsonized B. suis and B. suis manB (manB mutant) inside murine macrophages. Cells were pretreated with cholesterol-binding (filipin, 8 μg/ml) or -depleting (β-methyl cyclodextrin, 10 mM) molecules or the ganglioside GM1-binding molecule (cholera toxin B subunit, 40 μg/ml). Following treatment with the relevant drug, infection with Brucella spp., and postinfection incubation for 1 h, cells were lysed in Triton X-100 and intracellular viability at this short time (1 h) was measured as described in Materials and Methods. For each strain or condition, the control (100% value) was obtained in the absence of drug treatment. Experiments were performed in triplicate, and the values represent means ± standard deviations. The experiments were repeated at least four times.

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