Control of phagocytosis and phagosome maturation by phosphoinositides

Phagocytosis and phagosome maturation are integral elements of both the innate and adaptive immune systems. Recognition of specific moieties on the target particle by phagocytic receptors initiaties membrane and cytoskeletal remodeling that culminates with internalization of bound particle into a membrane-bound organelle. Rapidly, this nascent phagosome is remodeled, through interactions with endosomes and lysosomes, to a highly microbicidal compartment capable of eliminating the pathogen. This thesis examines the central role of phosphoinositide signaling in both of these processes. Subsequent to Fcgamma receptor activation, an actin-dense structure termed the phagocytic cup is assembled to facilitate the engulfment of the bound particle. Although much is known regarding the determinants of actin assembly at the phagocytic cup, the factors necessary for the disassembly of this actin structure are obscure. We therefore monitored several integral aspects of the forming phagosome, including the kinetics of actin assembly and remodeling, Rac1/Cdc42 activity, and phosphatidylinositol-4,5- bisphosphate (PI(4,5)P2) association with the forming phagosome to better understand the molecular processes involved in the later stages of phagocytosis. Interestingly, we found that PI(4,5)2 disappearance paralleled that of actin remodeling. To test the functional significance, we employed pharmacological and genetic approaches to demonstrate that hydrolysis of PI(4,5)P2 is requisite for actin remodeling at the phagocytic cup. Subsequent to entry into the cell, a distinct phosphoinositide, PI(3)P directs maturation. Herein, we demonstrate that PI(3)P association with the Salmonella-containing vacuole is necessary for vacuolar maturation. Further we demonstrate that in contrast to Salmonella, a separate intracellular pathogen, namely Mycobacteria, has decreased amounts of phagosomal PI(3)P. Together, these revelations implicate phosphoinositide signaling as essential for phagocytosis and phagosome maturation.