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. 2006 Nov 15;25(22):5250-9.
doi: 10.1038/sj.emboj.7601407. Epub 2006 Nov 2.

Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest

George B Kyei  1 , Isabelle Vergne, Jennifer Chua, Esteban Roberts, James Harris, Jagath R Junutula, Vojo Deretic
Affiliations

Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest

George B Kyei et al. EMBO J. .

Abstract

Mycobacterium tuberculosis arrests phagosomal maturation in infected macrophage, and, apart from health significance, provides a superb model system to dissect the phagolysosomal biogenesis pathway. Here, we demonstrate a critical role for the small GTPase Rab14 in maintaining mycobacterial phagosome maturation block. Four-dimensional microscopy showed that phagosomes containing live mycobacteria accumulated Rab14 following phagocytosis. The recruitment of Rab14 had strong functional consequence, as a knockdown of endogenous Rab14 by siRNA or overexpression of Rab14 dominant-negative mutants (Rab14S25N and Rab14N125I) released the maturation block and allowed phagosomes harboring live mycobacteria to progress into phagolysosomes. Conversely, overexpression of the wild-type Rab14 and the constitutively active mutant Rab14Q70L prevented phagosomes with dead mycobacteria from undergoing default maturation into phagolysosomal organelles. Mechanistic studies demonstrated a role for Rab14 in stimulating organellar fusion between phagosomes and early endosomes but not with late endosomes. Rab14 enables mycobacterial phagosomes to maintain early endosomal characteristics and avoid late endosomal/lysosomal degradative components.

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Figures

Figure 1
Figure 1
Differential recruitment of Rab14 to phagosomes harboring dead versus live mycobacteria. Macrophages were transfected with EGFP-Rab14WT, infected with Texas Red labeled BCG and imaging carried out by live confocal microscopy. (A) Number of live versus dead mycobacterial phagosomes recruiting EGFP-Rab14. Bars, means±s.e.m. (5 or more independent movies). A minimum of 30 phagosomes was counted for each condition. **P=0.01. (B) Ratiometric quantification over time of fluorescence intensity of EGFP-Rab14WT associated with phagosomes relative to fluorescence of the cytosol (RΦ/c). Shown are data from six independent movies (three each) of EGFP-Rab14 recruitment by live and dead BCG phagosomes. (C, D) Frames from time lapse live imaging show early recruitment of Rab14 by live mycobacteria (C) while dead mycobacteria (D) do not recruit Rab14. Displayed are maximum projections of confocal image stacks as previously described (Chua and Deretic, 2004). Insets: Left inset, gray-scale fluorescence of EGFP-Rab14 probe, right inset, gray-scale BCG fluorescence. Arrows indicate bacteria under observation.
Figure 2
Figure 2
Expression of dominant-negative Rab14 overcomes phagosome maturation block imposed by live mycobacteria. Macrophages were transfected with the indicated Rab14 constructs for 24 h, and infected with live Texas Red labeled BCG for 10 min. After 1 h, chase cells were fixed, stained for CD63 and examined by scanning confocal fluorescence microscopy. (A–U) Note colocalization between live BCG (red) and CD63 (blue) in cells transfected with Rab14S25N (M–P) and Rab14N125I (Q-T) as opposed to Rab14WT (E–H) and Rab14Q70L (I–L). (U) Quantification of colocalization between CD63 and live mycobacteria in cells transfected with Rab14 constructs. Bars, means±s.e.m. (3 independent experiments); n, number of phagosomes counted per condition. **P=0.007 (S25N), 0.0008 (N125I); †P=0.83 (Q70L), 0.77 (EGFP).
Figure 3
Figure 3
Expression of dominant-negative Rab14 promotes acquisition of V0-ATPase by phagosomes containing live mycobacteria. Macrophages transfected with the indicated Rab14 variants were pulsed with Texas Red labeled live BCG for 10 min. After 1 h chase cells were fixed, stained for Vo-ATPase and examined by confocal microscopy. (A–T) Note colocalization between BCG (red) and Vo-ATPase (blue) in cells transfected with Rab14S25N (M-P) and Rab14N125I (Q–T). (U) Quantification of colocalization between live mycobacteria and V0-ATPase. Bars, means±s.e.m. (3 independent experiments); n, number of phagosomes counted per condition. *P=0.01 (S25N), 0.042 (N125I); †P=0.36 (Q70L), 0.1 (EGFP).
Figure 4
Figure 4
Knockdown of Rab14 by siRNA promotes maturation of mycobacterial phagosomes. (A) Western blot of lysates from macrophages transfected with Rab14 siRNA or control (scrambled) siRNA. (B–C) RAW264.7 macrophages were transfected with Rab14 siRNA or control (scrambled) siRNA, infected with Texas Red labeled live BCG and phagosome maturation measured by colocalization between BCG and CD63 or Vo-ATPase. Note more frequent colocalization between live BCG (red) and CD63 (blue) or Vo-ATPase in Rab14 siRNA treated cells than in control siRNA treated cells. (D) U937 cells transfected with Rab14 siRNA for 48 h were infected with live M. tuberculosis H37Rv and phagosome maturation measured by accumulation of Lysotracker Red. Bars, means±s.e.m. (3 independent experiments); n, number of phagosomes counted. **P=0.0026 for CD63, 0.0012 for Vo-ATPase and 0.0005 for lysotracker red between Rab14 siRNA and controls.
Figure 5
Figure 5
Expression of constitutively active Rab14 inhibits maturation of phagosomes containing dead mycobacteria. RAW264.7 macrophages were transfected with Rab14 constructs or EGFP and infected with heat killed BCG. Colocalization between dead BCG and CD63 or Vo-ATPase was used to measure phagosome maturation. Note that dead mycobacteria in EGFP transfected cells show higher colocalization with CD63 or Vo-ATPase (A, E), compared to cells transfected with EGFP-Rab14Q70L (B, F) or EGFP-Rab14WT (C, G). Arrows in (E) indicate bacteria colocalizing with Vo-ATPase. (D, H) Quantification of CD63 and Vo-ATPase colocalization with BCG in cells transfected with EGFP or Rab14 variants. Bars, means±s.e.m. (3 independent experiments), n, number of phagosomes. **P=0.0037, ANOVA.
Figure 6
Figure 6
Rab14 promotes phagosomes-early endosome fusion in vitro. Phagosome–endosome fusion assay was carried out as described in Materials and methods, in the presence of cytosols prepared from untransfected 293T cells (none) or transfected with Rab14 variants. (A) Western blot showing expression of Rab14 fusion constructs in 293T cells with anti-EGFP antibody. (B) Phagosome–early endosome fusion. (C) Phagosome–late endosome fusion. (D) Western-blot showing levels of Rab14, Rab5 and GAPDH in cytosols used for fusion assay in (F). (E) Cytosol concentration-dependency of phagosome–early endosome fusion. (F) Phagosome–early endosome fusion in the presence of 2 mg/ml of cytosol from RAW264.7 cells transfected with scrambled siRNA (Control) or with Rab14 siRNA. Bars, means±s.e.m. (3 independent experiments). *P<0.05, **P<0.01 (t-test).
Figure 7
Figure 7
Rab14 promotes phagosome–phagosome fusion. (A) RAW264.7 cells were transfected with EGFP-Rab14WT, infected with live Texas Red labeled BCG and phagosomes monitored by live confocal microscopy. Panels show sequential images ending with fusion of two phagosomes that start out as separate phagosomes. (B) Transfected RAW264.7 macrophages were first infected with heat killed Texas Red labeled BCG, washed and subsequently infected with heat killed Alexa 647 labeled BCG (rendered in blue). Representative confocal images showing phagosome–phagosome fusion (arrow heads) in cells transfected with Rab14WT and Rab14Q70L. (C) Quantification of cells with at least one fused phagosome. Bars, mean±s.e.m. (3 independent experiments). A minimum of 50 doubly infected cells was counted for each condition. **P=0.02.
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References

    1. Allen LA, Schlesinger LS, Kang B (2000) Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages. J Exp Med 191: 115–128 - PMC - PubMed
    1. Armstrong JA, Hart PD (1971) Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J Exp Med 134: 713–740 - PMC - PubMed
    1. Barbero P, Bittova L, Pfeffer SR (2002) Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells. J Cell Biol 156: 511–518 - PMC - PubMed
    1. Braell WA (1992) Detection of endocytic vesicle fusion in vitro, using assay based on avidin–biotin association reaction. Methods Enzymol 219: 12–21 - PubMed
    1. Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M (1992) The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70: 715–728 - PubMed

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