PIKfyve is required for efficient phagosomal Rab7 acquisition and the delivery and fusion of early macropinosomes to...

Phagosome maturation is tightly regulated to ensure efficient delivery of the complex arsenal of antimicrobial activities that kill and digest captured microbes. Like other endocytic pathways, phagosome maturation is regulated by a combination of Rab GTPases and phosphoinositide (PIP) signalling lipids, which define membrane identity and recruit specific effectors. PIKfyve is a phosphoinositide 5-kinase, which converts phosphatidylinositol 3-phosphate [PI(3)P] into phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] on endosomes. Disruption of PIKfyve results in severe defects in phagosomal maturation but the underlying mechanism remains unclear. Here, we use the model professional phagocyte, Dictyostelium discoideum to dissect the role of PIKfyve in the crucial first steps of phagosome maturation. We find that, although early Rab5 dynamics are unaffected, loss of PIKfyve reduces phagosomal Rab7 acquisition by preventing fusion with a pool of Rab7 and V-ATPase positive endosomes. By following PIP dynamics using our recently characterised PI(3,5)P2 probe SnxA, we delineate multiple subpopulations of Rab7-positive endosomes that fuse sequentially with phagosomes. We identify one of these as PI(3,5)P2-positive macropinosomes, which dock and fuse with phagosomes in a PIKfyve-dependent manner. We therefore show that Dictyostelium phagosomes accumulate Rab7 by PIKfyve-dependent vesicular fusion in addition to acquisition from a cytosolic pool. We identity PI(3,5)P2-positive macropinosomes as a specific subset of fusogenic vesicles, which we propose enables content mixing and the efficient bulk delivery of lysosomal components to phagosomes.