Regulation of the macrophage vacuolar ATPase and phagosome-lysosome fusionby Histoplasma capsulatum

Histoplasma capsulatum (Hc) maintains a phagosomal pH of about 6.5. This st rategy allows He to obtain iron from transferrin, and minimize the activity of macrophage (Mo) lysosomal hydrolases. To determine the mechanism of pH regulation, we evaluated the function of the vacuolar ATPase (V-ATPase) in RAW264.7 Mo infected with Hc yeast or the nonpathogenic yeast Saccharomyces cerevisae (Sc), Incubation of Hc-infected MB with bafilomycin, an inhibito r of the V-ATPase, did not affect the intracellular growth of He, nor did i t affect the intraphagosomal pH, In contrast, upon addition of bafilomycin, phagosomes containing Sc rapidly changed their pH from 5 to 7, He-containi ng phagosomes had 5-fold less V-ATPase than Sc-containing phagosomes as qua ntified by immunoelectron microscopy, Furthermore, Hc-containing phagosomes inhibited phagolysosomal fusion as quantified by the presence of acid phos phatase, accumulation of LAMP2, and fusion with rhodamine B-isothiocyanate- labeled dextran-loaded lysosomes, Finally, in Hc-containing phagosomes, upt ake of ferritin was equivalent to phagosomes containing Sc, indicating that He-containing phagosomes have full access to the early "bulk flow"; endocy tic pathway. Thus, Hc yeasts inhibit phagolysosomal fusion, inhibit accumul ation of the V-ATPase in the phagosome, and actively acidify the phagosomal pH to 6.5 as part of their strategy to survive in Mo phagosomes.