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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 ofB. 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 liveB. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification ofB. suis -containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A1 , 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 ofB. suis , in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes withB. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage.

Early Acidification of Phagosomes ContainingBrucella suisIs Essential for Intracellular Survival in Murine Macrophages