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. 2006 Sep 25;174(7):923-9.
doi: 10.1083/jcb.200603026. Epub 2006 Sep 18.

Higher order Rab programming in phagolysosome biogenesis

Esteban A Roberts  1 , Jennifer Chua, George B Kyei, Vojo Deretic
Affiliations

Higher order Rab programming in phagolysosome biogenesis

Esteban A Roberts et al. J Cell Biol. .

Abstract

Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5-Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal maturation, the intracellular pathogen Mycobacterium tuberculosis prevents Rab7 acquisition, thus, providing a naturally occurring tool to study Rab conversion. We show that M. tuberculosis inhibition of Rab7 acquisition and arrest of phagosomal maturation depends on Rab22a. Four-dimensional microscopy revealed that phagosomes harboring live mycobacteria recruited and retained increasing amounts of Rab22a. Rab22a knockdown in macrophages via siRNA enhanced the maturation of phagosomes with live mycobacteria. Conversely, overexpression of the GTP-locked mutant Rab22aQ64L prevented maturation of phagosomes containing heat-killed mycobacteria, which normally progress into phagolysosomes. Moreover, Rab22a knockdown led to Rab7 acquisition by phagosomes harboring live mycobacteria. Our findings show that Rab22a defines the critical checkpoint for Rab7 conversion on phagosomes, allowing or disallowing organellar transition into a late endosomal compartment. M. tuberculosis parasitizes this process by actively recruiting and maintaining Rab22a on its phagosome, thus, preventing Rab7 acquisition and blocking phagolysosomal biogenesis.

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Figures

Figure 1.
Figure 1.
Dynamics of group V Rabs on phagosomes. RAW264.7 transfected with EGFP-Rab5WT (A and D), EGFP-Rab21WT (B and E), and EGFP-Rab22WT (C and F) were allowed to phagocytose Texas red–labeled latex beads or Texas red–labeled live M. tuberculosis variant bovis BCG. EGFP-Rab5WT (A), EGFP-Rab21WT (B), and EGFP-Rab22aWT (C) were transiently recruited to latex bead phagosomes and subsequently dissociated. EGFP-Rab5WT (D) and EGFP-Rab21WT (E) are recruited transiently on mycobacterial phagosomes and immediately dissociate. In contrast, EGFP-Rab22aWT was retained on mycobacterial phagosomes throughout the imaging period (F) of 1 h after phagocytosis. Left insets correspond to GFP fluorescence of the Rabs and the right insets correspond to the fluorescence of latex beads or mycobacteria. All microscopy imaging was performed using an UltraView LCI system. (G–I) Temporal quantification of phagosome fluorescence intensity relative to fluorescence of the cytosol. RΦ/C, ratio between phagosome fluorescence intensity and cytosol fluorescence intensity. Shown are RΦ/C obtained by 4D microscopy and live imaging of EGFP-Rab5WT (G), EGFP-Rab21WT (H), and EGFP-Rab22aWT (I) harboring latex beads (open triangles) and live mycobacteria (filled squares). The number of phagosomes counted are as follows: EGFP-Rab5WT = 35; EGFP-Rab21W T = 24; EGFP-Rab22aWT = 47. (J) Quantification of EGFP-Rab5WT, EGFP-Rab21WT, and EGFP-Rab22aWT positivity on the latex bead phagosomes (open bars) and live mycobacterial phagosomes (filled bars) during the first 10 min after phagocytosis. *, P = 0.0002. (K) Quantification of EGFP-Rab5WT, EGFP-Rab21WT, and EGFP-Rab22aWT positivity on the latex bead phagosomes (open bars) and live mycobacterial phagosomes (filled bars) 45 min after phagocytosis. *, P < 0.0001.
Figure 2.
Figure 2.
Constitutively active Rab22aQ64L prevents normal maturation of phagosomes harboring dead mycobacterial phagosomes. RAW264.7 cells were transfected with EGFP-Rab22aQ64L and infected with dead mycobacteria in the presence of Lysotracker Blue (A–E) or DQ Red BSA (G–K). A and G show fluorescence from GFP. B and H show the fluorescence of dead mycobacteria labeled with Texas red and Alexa Fluor 647, respectively. C and I show the fluorescence of Lysotracker Blue (blue) and DQ Red BSA (red), respectively. D shows the merge image of mycobacteria (red) and Lysotracker Blue (blue). J shows the merge image of mycobacteria (blue) and DQ Red BSA (red). E and K represent RGB composite images. Arrows indicate no colocalization between dead mycobacterial phagosomes and probes in EGFP-Rab22aQ64L–expressing cells. (F) Quantification of Lysotracker Blue staining on dead mycobacterial phagosomes in control versus EGFP-Rab22aQ64L–expressing cells. P = 0.0001. (L) Quantification of DQ Red BSA staining on dead mycobacterial phagosomes in control versus EGFP-Rab22aQ64L–expressing cells. *, P < 0.0001.
Figure 3.
Figure 3.
EGFP-Rab22aQ64L confers recycling endosomal characteristics on dead mycobacterial phagosomes. Nontransfected RAW264.7 cells (A–D and I–L) and EGFP-Rab22aQ64L–expressing cells (E–H and M–P) were infected with dead mycobacteria and immunostained for TfR (A–H) and syntaxin 13 (I–P). Anti-TfR antibodies did not stain dead mycobacterial phagosomes (A–D) in control cells, whereas staining can be seen in EGFP-Rab22aQ64L–expressing cells (E–H). Anti-syntaxin 13 antibodies also did not stain dead mycobacterial phagosomes in control cells (I–L), but stained in EGFP-Rab22aQ64L–transfected cells (M–P). (Q) Quantification of TfR-positive dead mycobacterial phagosomes. *, P = 0.0063. (R) Quantification of syntaxin13–positive dead mycobacterial phagosomes. *, P < 0.0001.
Figure 4.
Figure 4.
Rab22a knockdown promotes the maturation of phagosomes with live mycobacteria. RAW264.7 macrophages were transfected with siRNA to Rab22a for 24 h and lysates probed for endogenous Rab22a knockdown via immunoblotting (A). There was no observable effect on the levels of TfR by Rab22a knockdown (A). RAW264.7 macrophages were transfected with siRNA to Rab22a for 24 h to provide sufficient suppression of Rab22a expression and subsequently infected with Texas red–labeled live M. tuberculosis variant bovis BCG for 10 min, followed by a 1-h chase period. Although only 13% of live mycobacterial phagosomes colocalize with CD63-positive compartments in macrophages transfected with scrambled siRNA (B and E), 40% of live mycobacterial phagosomes were positive for CD63 colocalization in cells transfected with Rab22a siRNA (B and E). Similarly, Rab22a siRNA–mediated inhibition enhanced the colocalization of live mycobacterial phagosomes with V0-positive compartments (C and F) by 2.8-fold when compared with minimal phagosomal colocalization in scrambled siRNA–transfected cells (C and F). Rab22a siRNA significantly inhibited the colocalization of Texas red–labeled live M. tuberculosis variant bovis BCG phagosomes with TfR (D and G) compared with scrambled siRNA (D and G). These results demonstrate that Rab22a is essential for mycobacterial ability in maintaining an immature phagosomal niche.
Figure 5.
Figure 5.
Rab7 acquisition by phagosomes is dependent on Rab22a. Rab22a knockdown in RAW264.7 macrophages led to increased late endosomal GTPase Rab7 recruitment to live mycobacterial phagosomes (D–F and G) compared with macrophages transfected with scrambled siRNA (A–C and G).
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