Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular sig nificance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes a re not completely understood. Here, we applied the property of pathogenic m ycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the r equirement for 3-phosphoinositides and acquisition of Rab5 effector early e ndosome autoantigen (EEA1) as essential molecular events necessary for phag osomal maturation. Unlike the model phagosomes containing latex beads, whic h transiently recruited EEA1, mycobacterial phagosomes excluded this regula tor of vesicular trafficking that controls membrane tethering and fusion pr ocesses within the endosomal pathway and is recruited to endosomal membrane s via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recr uitment to newly formed latex bead phagosomes and blocked phagosomal acquis ition of late endocytic properties, indicating that generation of PtdIns(3) P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late e ndocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exc lusion from mycobacterial phagosomes was investigated using mycobacterial p roducts. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virul ent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to late x bead phagosomes, and diminished their maturation. These findings define t he generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical m olecular targets affected by M. tuberculosis. This study also identifies my cobacterial phosphoinositides as products with specialized toxic properties , interfering with discrete trafficking stages in phagosomal maturation.