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© The Rockefeller University Press, 0021-9525/2001/8/631 $5.00
The Journal of Cell Biology, Volume 154, Number 3, August 6, 2001 631-644

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

¹ Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109
² Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461
³ Department of Biochemistry, University of Geneva, CH-1211 Geneva, Switzerland
⁴ Department of Molecular Medicine and Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655
⁵ Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI 48109

Address correspondence to Vojo Deretic, University of Michigan Medical School, 5641 Medical Science Bldg. II, Ann Arbor, MI 48109-0620. Tel.: (734) 763-1580. Fax: (734) 647-6243. E-mail: deretic{at}umich.edu

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition 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 endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.

Key Words: EEA1; endosome; hVPS34; LBPA; LAM

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