Figure 5.
CHC22 mediates membrane traffic from the ERGIC to the L.p. replicative vacuole, which acquires some GLUT4 pathway markers. (A) Representative images of L.p.-infected A549 cells transiently transfected with GFP-tagged CHC22 or CHC17 (green). 1 h after infection with either WT or mutant ΔdotA L.p. (MOI = 50), bacteria were detected by IF (red). Arrowheads point to L.p., and boxed inserts (upper right or left) show the L.p. region at 5× magnification. Scale bars: 10 µm for cells expressing CHC22-GFP; 7.5 µm for cells expressing CHC17-GFP. (B) Quantification of the proportion of L.p. vacuoles positive for CHC22 or CHC17. Data expressed as mean ± SEM, n = 3, 4–35 vacuoles counted per experiment performed as represented in A. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test, ***, P < 0.001. (C) Representative images of A549 cells infected with WT L.p. (MOI = 50) immunolabeled for endogenous CHC22 or CHC17 (red) and L.p. (green) by IF. Arrowheads point to L.p.; dashed lines delineate cell borders. Scale bars: 5 µm. (D) Quantification of the proportion of replicative vacuoles (8 h after infection) containing one, two to four, or more than four WT or ΔdotA L.p. after treatment with siRNA targeting CHC22 (siCHC22) or CHC17 (siCHC17) or nontargeting siRNA (siControl). Data expressed as mean ± SEM, n = 3, >140 vacuoles counted per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test was performed to compare the number of cells with a vacuole containing more than four bacteria. ****, P < 0.0001 versus siControl-transfected cells infected with WT L.p.++++, P < 0.0001 versus siControl-transfected cells infected with ΔdotA L.p.(E) Quantification of the proportion of L.p. vacuoles positive for GLUT4-GFP, p115, GGA2, sortilin, IRAP, or Rab1 1 h after infection with WT or ΔdotA L.p. in HeLa cells transiently expressing FcγRII (needed for L.p. infection; Arasaki and Roy, 2010). Data expressed as mean ± SEM, n = 3, 4–50 vacuoles counted per experiment. Two-tailed unpaired Student’s t test with equal variances: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F–I) Representative IF of HeLa cells 1 h after infection with either WT or mutant ΔdotA L.p. (MOI = 50) stained for L.p. (red) and sortilin (F), IRAP (G), GGA2 (H), or p115 (green; I). Hoechst stains the nuclei blue. Arrows point to LCVs. Scale bars: 10 µm. Merged images in A, C, and F–I show red/green overlap in yellow.

CHC22 mediates membrane traffic from the ERGIC to the L.p. replicative vacuole, which acquires some GLUT4 pathway markers. (A) Representative images of L.p.-infected A549 cells transiently transfected with GFP-tagged CHC22 or CHC17 (green). 1 h after infection with either WT or mutant ΔdotA L.p. (MOI = 50), bacteria were detected by IF (red). Arrowheads point to L.p., and boxed inserts (upper right or left) show the L.p. region at 5× magnification. Scale bars: 10 µm for cells expressing CHC22-GFP; 7.5 µm for cells expressing CHC17-GFP. (B) Quantification of the proportion of L.p. vacuoles positive for CHC22 or CHC17. Data expressed as mean ± SEM, n = 3, 4–35 vacuoles counted per experiment performed as represented in A. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test, ***, P < 0.001. (C) Representative images of A549 cells infected with WT L.p. (MOI = 50) immunolabeled for endogenous CHC22 or CHC17 (red) and L.p. (green) by IF. Arrowheads point to L.p.; dashed lines delineate cell borders. Scale bars: 5 µm. (D) Quantification of the proportion of replicative vacuoles (8 h after infection) containing one, two to four, or more than four WT or ΔdotA L.p. after treatment with siRNA targeting CHC22 (siCHC22) or CHC17 (siCHC17) or nontargeting siRNA (siControl). Data expressed as mean ± SEM, n = 3, >140 vacuoles counted per experiment. One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test was performed to compare the number of cells with a vacuole containing more than four bacteria. ****, P < 0.0001 versus siControl-transfected cells infected with WT L.p.++++, P < 0.0001 versus siControl-transfected cells infected with ΔdotA L.p.(E) Quantification of the proportion of L.p. vacuoles positive for GLUT4-GFP, p115, GGA2, sortilin, IRAP, or Rab1 1 h after infection with WT or ΔdotA L.p. in HeLa cells transiently expressing FcγRII (needed for L.p. infection; Arasaki and Roy, 2010). Data expressed as mean ± SEM, n = 3, 4–50 vacuoles counted per experiment. Two-tailed unpaired Student’s t test with equal variances: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F–I) Representative IF of HeLa cells 1 h after infection with either WT or mutant ΔdotA L.p. (MOI = 50) stained for L.p. (red) and sortilin (F), IRAP (G), GGA2 (H), or p115 (green; I). Hoechst stains the nuclei blue. Arrows point to LCVs. Scale bars: 10 µm. Merged images in A, C, and F–I show red/green overlap in yellow.

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