Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages - PubMed (original) (raw)
Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages
F Porte et al. Infect Immun. 1999 Aug.
Abstract
Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication of B. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing live B. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification of B. suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A(1), a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes with B. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.
Figures
FIG. 1
Intracellular survival of B. suis 1330 in murine J774 cells. Macrophages were infected with unlabeled (●) and with CF-Rho-labeled (○), opsonized bacteria as described in the text. Each point represents the mean ± standard deviation of three experiments.
FIG. 2
Acidification of phagosomes containing live B. suis labeled with CF and Rho. Bacteria were labeled with fluorescent probes and then opsonized with specific antibodies. (A) In situ calibration curve for the calculation of phagosomal pH from the CF/Rho emission ratio. Cells infected with live B. suis were incubated in buffers of defined pH containing the ionophore nigericin. The plotted emission ratios represent the means ± standard deviations calculated from the acquisition of four pairs of images for each pH value. (B) Phagosomal pH after infection with live bacteria. At the times indicated, ranging from 1 to 5 h postinfection, four pairs of CF and Rho images were acquired from every observation, and pH was calculated from the calibration curve. Experiments were done in quadruplicate.
FIG. 3
Acidification of phagosomes containing live B. suis labeled with Oregon Green 488 and Rho. Bacteria were labeled with fluorescent probes and then opsonized with specific antibodies. (A) In situ calibration curve for the calculation of phagosomal pH from the Oregon Green/Rho emission ratio. Cells infected with live B. suis were incubated in buffers of defined pH containing the ionophore nigericin. The plotted emission ratios, represent the means ± standard deviations calculated from the acquisition of four pairs of images for each pH value. (B) Phagosomal pH after infection with live bacteria. At the times indicated, ranging from 2 to 5 h postinfection, four pairs of Oregon green 488 and Rho images were acquired from every observation, and pH was calculated from the calibration curve.
FIG. 4
Effect of bafilomycin A1 on acidification of phagosomes containing live or gentamicin-killed B. suis. (A) Phagosomal pH after infection with live bacteria, in the absence (open bars) or presence (grey bars) of 100 nM bafilomycin A1, added at the end of the infection period. Live bacteria were labeled with fluorescent probes (CF and Rho) and then opsonized with specific antibodies. (B) Phagosomal pH after infection with killed bacteria, in the absence (open bars) or presence (grey bars) of 100 nM bafilomycin A1. Bacteria were first gentamicin killed, labeled with fluorescent probes (CF and Rho), and then opsonized with specific antibodies. Calibration curves were made for each experiment. Values represent means ± standard deviations.
FIG. 5
Effects of vacuolar-pH-neutralizing reagents on survival of opsonized (A) or nonopsonized (B) B. suis in macrophages. Bafilomycin (BAF; 100 nM), NH4Cl (30 mM), or monensin (MON; 50 μM) was added to J774 cells 1 h postinfection. The number of surviving bacteria was determined 3 and 5 h later (4 and 6 h postinfection [p.i.]). Percentages are indicated with respect to the number of viable bacteria at 1 h, prior to addition of the reagents, considered to be 100%. NT, no treatment (control). All experiments were performed in quadruplicate. Values represent means ± standard deviations.
FIG. 6
Time-dependent effect of the neutralization of cellular compartments by 30 mM NH4Cl on intracellular survival of B. suis 1330 in murine J774 cells. During the course of infection, macrophages remained untreated (●) or were treated with NH4Cl at 90 min (▾) or 7 h (○) after the beginning of infection. Experiments were performed in triplicate, and the values represent means ± standard deviations.
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