p110-related PI 3-kinases regulate phagosome-phagosome fusion and phagosomal pH through a PKB/Akt dependent pathway in Dictyostelium

The Dictyostelium p110-related PI 3-kinases, PIK1 and PIK2, regulate the en dosomal pathway and the actin cytoskeleton, but do not significantly regula te internalization of particles in D, discoideum, Bacteria internalized int o Delta ddpik1/ddpik2 cells or cells treated with BI 3-kinase inhibitors re mained intact as single particles in phagosomes with closely associated mem branes after 2 hours of internalization, while in control cells, bacteria a ppeared degraded in multi-particle spacious phagosomes. Addition of LY29400 2 to control cells, after 60 minutes of chase, blocked formation of spaciou s phagosomes, suggesting PI 3-kinases acted late to regulate spacious phago some formation, Phagosomes purified from control and drug treated cells con tained equivalent levels of lysosomal proteins, including the proton pump c omplex, and were acidic, but in drug treated cells and Delta ddpik1/ddpik2 cells phagosomal pH was significantly more acidic during maturation than th e pH of control phagosomes. Inhibition of phagosomal maturation by LY294002 was overcome by increasing phagosomal pH with NH4Cl, suggesting that an in crease in pH might trigger homotypic phagosome fusion, A pkbA null cell lin e (PKB/Akt) reproduced the phenotype described for cells treated with PI 3- kinase inhibitors and Delta ddpik1/ddpik2 cells. We propose that PI 3-kinas es, through a PKB/Akt dependent pathway, directly regulate homotypic fusion of single particle containing phagosomes to form multiparticle, spacious p hagosomes, possibly through the regulation of phagosomal pH.