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

Fratti RA, Backer JM, Gruenberg J, Corvera S, Deretic V - J. Cell Biol. (2001)

Bottom Line: We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

EEA1 exclusion reduces LBPA accumulation on latex beads. (A) Quantitation of LBPA colocalization with latex bead phagosomes (30 min after infection) in cells injected with anti-hVPS34, anti-EEA1, control rabbit IgG, and uninjected control cells (n = 1,347 phagosomes). The data are means ± SE of three separate experiments. *P = 0.0001. (B) Quantitation of EEA1 colocalization with latex bead phagosomes (30 min after infection) in cells injected with anti-hVPS34, control rabbit IgG, and uninjected control cells (n = 601 phagosomes). The data are means ± SE of three separate experiments. *P < 0.001. (C) M. tuberculosis ManLAM but not its precursor PIM reduces EEA1 recruitment to the phagosome. Epifluorescence microscopy analysis of the effect of coating of latex beads with mycobacterial phosphoinositides on EEA1 recruitment. Latex beads were coated with purified M. tuberculosis H37Rv ManLAM or PIM and phagocytosed by J774 cells for 20 min. Quantitation of EEA1 colocalization with phagosomes (n = 1,321 phagosomes). Black bars, control or lipid-coated beads (as indicated) not treated with Triton X-100; hatched bars, control or lipid-coated beads (as indicated) extracted with Triton X-100 before infection. The data are means ± SE of three separate experiments. **P = 0.002. M. tuberculosis glycosylated phosphoinositides do not affect Syntaxin 8 acquisition by latex bead phagosomes. Epifluorescence microscopy analysis of Syntaxin 8 accumulation by phagosomes containing control uncoated latex beads or beads coated with mycobacterial phosphoinositides. Latex beads were coated with ManLAM or PIM and phagocytosed by J774 cells for 20 min. White bars, quantitation of Syntaxin 8 colocalization with phagosomes (n = 432 phagosomes). The data are means ± SE of three separate experiments. (D) M. tuberculosis ManLAM reduces LBPA recruitment to the phagosome. Epifluorescence microscopy analysis of the effect of coating of latex beads with ManLAM on LBPA accumulation. Quantitation of LBPA colocalization with phagosomes after 30 min of phagocytosis (n = 506 phagosomes). The data are means ± SE of three separate experiments. *P = 0.0001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196432&req=5

fig7: EEA1 exclusion reduces LBPA accumulation on latex beads. (A) Quantitation of LBPA colocalization with latex bead phagosomes (30 min after infection) in cells injected with anti-hVPS34, anti-EEA1, control rabbit IgG, and uninjected control cells (n = 1,347 phagosomes). The data are means ± SE of three separate experiments. *P = 0.0001. (B) Quantitation of EEA1 colocalization with latex bead phagosomes (30 min after infection) in cells injected with anti-hVPS34, control rabbit IgG, and uninjected control cells (n = 601 phagosomes). The data are means ± SE of three separate experiments. *P < 0.001. (C) M. tuberculosis ManLAM but not its precursor PIM reduces EEA1 recruitment to the phagosome. Epifluorescence microscopy analysis of the effect of coating of latex beads with mycobacterial phosphoinositides on EEA1 recruitment. Latex beads were coated with purified M. tuberculosis H37Rv ManLAM or PIM and phagocytosed by J774 cells for 20 min. Quantitation of EEA1 colocalization with phagosomes (n = 1,321 phagosomes). Black bars, control or lipid-coated beads (as indicated) not treated with Triton X-100; hatched bars, control or lipid-coated beads (as indicated) extracted with Triton X-100 before infection. The data are means ± SE of three separate experiments. **P = 0.002. M. tuberculosis glycosylated phosphoinositides do not affect Syntaxin 8 acquisition by latex bead phagosomes. Epifluorescence microscopy analysis of Syntaxin 8 accumulation by phagosomes containing control uncoated latex beads or beads coated with mycobacterial phosphoinositides. Latex beads were coated with ManLAM or PIM and phagocytosed by J774 cells for 20 min. White bars, quantitation of Syntaxin 8 colocalization with phagosomes (n = 432 phagosomes). The data are means ± SE of three separate experiments. (D) M. tuberculosis ManLAM reduces LBPA recruitment to the phagosome. Epifluorescence microscopy analysis of the effect of coating of latex beads with ManLAM on LBPA accumulation. Quantitation of LBPA colocalization with phagosomes after 30 min of phagocytosis (n = 506 phagosomes). The data are means ± SE of three separate experiments. *P = 0.0001.

Mentions: To determine whether hVPS34 recruitment to phagosomal membranes is required for phagosome maturation, hVPS34 was inhibited by microinjecting an isoform-specific inhibitory antibody against this Rab5 effector (Siddhanta et al., 1998). Phagosome maturation was assessed by accumulation of LBPA. When the cells were injected with anti-hVPS34 antibody, latex bead phagosomes manifested a markedly reduced accumulation of LBPA relative to phagosomes in control cells. Although >58% of latex bead phagosomes in the control acquired LBPA at 30 min after phagocytosis (Fig. 6 I and Fig. 7 A), only 15% of phagosomes in cells injected with anti-hVPS34 antibody accumulated LBPA (Fig. 6 J and Fig. 7 A) (P = 0.0001; ANOVA). Microinjecting cells with control rabbit IgG did not affect LBPA accumulation (P = 0.41; ANOVA) (Fig. 6 L and Fig. 7 A). These data demonstrate that hVPS34 plays a role in phagosome maturation into vacuoles with late endosomal characteristics. One possible explanation for the maturation-arresting effect of microinjected anti-hVPS34 antibody is that it may prevent or reduce EEA1 recruitment. To examine this possibility, we tested the effect of anti-hVPS34 antibody on EEA1 recruitment to phagosomes. EEA1 localization to latex bead phagosomes was reduced by 63% in cells injected with anti-hVPS34 relative to uninjected cells or cells injected with control rabbit IgG (P < 0.001; ANOVA) (Fig 7 B).


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

Fratti RA, Backer JM, Gruenberg J, Corvera S, Deretic V - J. Cell Biol. (2001)

EEA1 exclusion reduces LBPA accumulation on latex beads. (A) Quantitation of LBPA colocalization with latex bead phagosomes (30 min after infection) in cells injected with anti-hVPS34, anti-EEA1, control rabbit IgG, and uninjected control cells (n = 1,347 phagosomes). The data are means ± SE of three separate experiments. *P = 0.0001. (B) Quantitation of EEA1 colocalization with latex bead phagosomes (30 min after infection) in cells injected with anti-hVPS34, control rabbit IgG, and uninjected control cells (n = 601 phagosomes). The data are means ± SE of three separate experiments. *P < 0.001. (C) M. tuberculosis ManLAM but not its precursor PIM reduces EEA1 recruitment to the phagosome. Epifluorescence microscopy analysis of the effect of coating of latex beads with mycobacterial phosphoinositides on EEA1 recruitment. Latex beads were coated with purified M. tuberculosis H37Rv ManLAM or PIM and phagocytosed by J774 cells for 20 min. Quantitation of EEA1 colocalization with phagosomes (n = 1,321 phagosomes). Black bars, control or lipid-coated beads (as indicated) not treated with Triton X-100; hatched bars, control or lipid-coated beads (as indicated) extracted with Triton X-100 before infection. The data are means ± SE of three separate experiments. **P = 0.002. M. tuberculosis glycosylated phosphoinositides do not affect Syntaxin 8 acquisition by latex bead phagosomes. Epifluorescence microscopy analysis of Syntaxin 8 accumulation by phagosomes containing control uncoated latex beads or beads coated with mycobacterial phosphoinositides. Latex beads were coated with ManLAM or PIM and phagocytosed by J774 cells for 20 min. White bars, quantitation of Syntaxin 8 colocalization with phagosomes (n = 432 phagosomes). The data are means ± SE of three separate experiments. (D) M. tuberculosis ManLAM reduces LBPA recruitment to the phagosome. Epifluorescence microscopy analysis of the effect of coating of latex beads with ManLAM on LBPA accumulation. Quantitation of LBPA colocalization with phagosomes after 30 min of phagocytosis (n = 506 phagosomes). The data are means ± SE of three separate experiments. *P = 0.0001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196432&req=5

fig7: EEA1 exclusion reduces LBPA accumulation on latex beads. (A) Quantitation of LBPA colocalization with latex bead phagosomes (30 min after infection) in cells injected with anti-hVPS34, anti-EEA1, control rabbit IgG, and uninjected control cells (n = 1,347 phagosomes). The data are means ± SE of three separate experiments. *P = 0.0001. (B) Quantitation of EEA1 colocalization with latex bead phagosomes (30 min after infection) in cells injected with anti-hVPS34, control rabbit IgG, and uninjected control cells (n = 601 phagosomes). The data are means ± SE of three separate experiments. *P < 0.001. (C) M. tuberculosis ManLAM but not its precursor PIM reduces EEA1 recruitment to the phagosome. Epifluorescence microscopy analysis of the effect of coating of latex beads with mycobacterial phosphoinositides on EEA1 recruitment. Latex beads were coated with purified M. tuberculosis H37Rv ManLAM or PIM and phagocytosed by J774 cells for 20 min. Quantitation of EEA1 colocalization with phagosomes (n = 1,321 phagosomes). Black bars, control or lipid-coated beads (as indicated) not treated with Triton X-100; hatched bars, control or lipid-coated beads (as indicated) extracted with Triton X-100 before infection. The data are means ± SE of three separate experiments. **P = 0.002. M. tuberculosis glycosylated phosphoinositides do not affect Syntaxin 8 acquisition by latex bead phagosomes. Epifluorescence microscopy analysis of Syntaxin 8 accumulation by phagosomes containing control uncoated latex beads or beads coated with mycobacterial phosphoinositides. Latex beads were coated with ManLAM or PIM and phagocytosed by J774 cells for 20 min. White bars, quantitation of Syntaxin 8 colocalization with phagosomes (n = 432 phagosomes). The data are means ± SE of three separate experiments. (D) M. tuberculosis ManLAM reduces LBPA recruitment to the phagosome. Epifluorescence microscopy analysis of the effect of coating of latex beads with ManLAM on LBPA accumulation. Quantitation of LBPA colocalization with phagosomes after 30 min of phagocytosis (n = 506 phagosomes). The data are means ± SE of three separate experiments. *P = 0.0001.
Mentions: To determine whether hVPS34 recruitment to phagosomal membranes is required for phagosome maturation, hVPS34 was inhibited by microinjecting an isoform-specific inhibitory antibody against this Rab5 effector (Siddhanta et al., 1998). Phagosome maturation was assessed by accumulation of LBPA. When the cells were injected with anti-hVPS34 antibody, latex bead phagosomes manifested a markedly reduced accumulation of LBPA relative to phagosomes in control cells. Although >58% of latex bead phagosomes in the control acquired LBPA at 30 min after phagocytosis (Fig. 6 I and Fig. 7 A), only 15% of phagosomes in cells injected with anti-hVPS34 antibody accumulated LBPA (Fig. 6 J and Fig. 7 A) (P = 0.0001; ANOVA). Microinjecting cells with control rabbit IgG did not affect LBPA accumulation (P = 0.41; ANOVA) (Fig. 6 L and Fig. 7 A). These data demonstrate that hVPS34 plays a role in phagosome maturation into vacuoles with late endosomal characteristics. One possible explanation for the maturation-arresting effect of microinjected anti-hVPS34 antibody is that it may prevent or reduce EEA1 recruitment. To examine this possibility, we tested the effect of anti-hVPS34 antibody on EEA1 recruitment to phagosomes. EEA1 localization to latex bead phagosomes was reduced by 63% in cells injected with anti-hVPS34 relative to uninjected cells or cells injected with control rabbit IgG (P < 0.001; ANOVA) (Fig 7 B).

Bottom Line: We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus