Phagocytosis of microorganisms by leukocytes represents an important first line of define in the prevention of systemic infections. Phagosomes containing pathogenic organisms are usually modified by a series of fusion and fission reactions, involving compartments of the endo-lysosomal system, which results in the destruction of the organism in the hostile milieu of the phago-lysosome. Many pathogens, including the causative effect of tuberculosis (Mycobacteria), have evolved mechanisms to prevent the maturations of phagosomes to phago-lysosomes, thus allowing for their survival. Little is known concerning the biochemical mechanisms that regulate the fission and fusion reactions regulation phagosome maturation, although it has been hypothesized that the Rab family of small G proteins are phosphatidylinositide 3-kinases (PI 3-kinases) play an important role. Given the significant increase world-wide in the incidence of infectious diseases, it is critical to understand the biochemical mechanisms regulating phagocytosis maturation in order to develop ratione therapeutic approaches. The mechanism regulating phagocytosis are currently being investigated in the genetically tractable amoeba, Dictyostelium discoideum. These studies have revealed at least 4 stages in phagosomal maturation, including the formation of spacious phagosomes containing multiple particles, a process exploited by many intracellular pathogens. Genetic and biochemical approaches have been used to determine that a GPTase, Rab7, and the PI 3- kinases, DdPIK1 and DdPIK2, appear to regulated this process. Furthermore, a novel Rab, RabB, also found in humans cells, regulated phagocytosis. To determine the biochemical mechanisms that operate to regulate phagosomal maturation, the following specific aims are proposed.
In specific aim#1, genetic and biochemical approaches will be used to: 1) determine if PI 3- kinases act directly by phosphorylating lipids in the phagosome membrane to regulate homotypic fusion; 2) determine if Rab7 and PI 3-kinases are functionally coupled and 3) identify Rab7 effector proteins.
In specific aim #2, the biochemical mechanisms of action of RabB in regulating phagocytosis and phagosomal maturation will be better defined. The results from these studies should supply answers to the following important questions: 1) do Rab7 and PI 3-kinase functionally and physically interact: 2) do PI-kinases and Rab7 regulated other steps in the phagosomal maturation pathway, in addition to homotypic fusion: 3) what is the biochemical nature of the effector proteins that interact with Rab7; 4) how does RabB regulate internalization of particles and 5) how does RabB function elsewhere in the phagosomal pathway?