Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages

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 su rvival and multiplication of B. suis. Intraphagosomal pH in murine J774 cel ls was determined by measuring the fluorescence intensity of opsonized, car boxyfluorescein-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 ba filomycin A(1), a specific inhibitor of vacuolar proton-ATPases. This neutr alization at 1 h postinfection resulted in a 2- to 34-fold reduction of ops onized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfe ction, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of am monium chloride at 7 h after the beginning of infection, however, did not a ffect intracellular multiplication of B. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. T hus, we conclude that phagosomes with B. suis acidify rapidly after infecti on, and that this early acidification is essential for replication of the b acteria within the macrophage.