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cmi12087-sup-0001-si.tif1355K

Fig. S1. Vamp8 does not colocalize with Vamp7 in the fully developed CRVs.

A. Transiently transfected HeLa cells overexpressing pEGFP-Vamp8 were infected with C. burnetii. After 48 h of infection, the cells were fixed and subjected to a double indirect immunofluorescence using specific antibodies against Vamp7 (red) and C. burnetii (blue). The cells were analysed by confocal microscopy. Insets show no colocalization between endogenous Vamp7 and overexpressed Vamp8 on the Coxiella vacuole (see arrows).

B. HeLa cells were infected with Coxiella burnetii. After infection, the cells were fixed at different times (1 h, 6 h, 12 h 24 h and 48 h) and subjected to a double immunofluorescence using specific antibodies against C. burnetii (red) and Syntaxin1 (Stx1, green). The images show no-colocalization between C. burnetii and Stx1 at any tested times.

cmi12087-sup-0002-si.tif506K

Fig. S2. Vti1a and Vti1b are recruited to the Coxiella phagosomes in a bacterial-dependent manner. Coxiella burnetii was pretreated with 100 μg ml−1 chloramphenicol for 30 min. After treatment, HeLa cells were infected with either control or chloramphenicol-pretreated Coxiella, maintaining the antibiotic in the culture medium, and fixed at different infection times (1 h, 6 h, 12 h and 24 h). After fixation the cells were subjected to a double immunofluorescence using specific antibodies against C. burnetii and Vti1a or Vti1b.

A. The bar graph shows that in cells that were treated with chloramphenicol the colocalization between endogenous Vti1a and Coxiella decreased significantly at all times analysed.

B. The bar graph shows that in cells that were treated with chloramphenicol the colocalization between endogenous Vti1a and Coxiella decreased significantly at all times analysed.

cmi12087-sup-0003-si.tif306K

Fig. S3. Overexpression of EGFP-Vamp7 wt or EGFP-Vamp7 NT does not affect Coxiella internalization in HeLa cells. HeLa cells were transfected with pEGFP-Vamp7 wt or pEGFP-Vamp7 NT. After 24 h of transfection the cells were infected with C. burnetii for 2 h in a multiplicity of infection of 10 (A) or 20 (B), fixed and subjected to indirect immunofluorescence using a specific antibody against C. burnetii.

cmi12087-sup-0004-si.tif285K

Fig. S4. Overexpression of the tetanus toxin does not affect the homotypic and heterotypic fusion capacity of the CRVs.

A. HeLa cells transiently transfected with EGFP-Vamp3 wt or cells transiently co-transfected with EGFP-Vamp3 wt and TeNT were infected with Coxiella burnetii. At 2 h of infection, cells were fixed and subjected to a double immunofluorescence using specific antibodies against CD63 (LAMP 3) and C. burnetii. The cells were analysed by confocal microscopy. Quantification of the colocalization between C. burnetii and CD63 shows a non-significant decrease in the percentage of colocalization in cells overexpressing the tetanus toxin compared with the control condition. Data represent the mean ± SEM of at least two independent experiments.

B. HeLa cells were infected with C. burnetii for 24 h and transfected with pEGFP-Vamp7 wt or pEGFP-Vamp7 NT. After 24 h of transfection, cells were infected with mCherry Coxiella for 2 h, fixed and subjected to indirect immunofluorescence using specific antibodies against C. burnetii. The cells were analysed by confocal microscopy. Quantification of the colocalization between C. burnetii and mCherry-Coxiella. The bar graph shows non-significant differences in the percentage of colocalization under TeNT overexpression compared with the control.

cmi12087-sup-0005-si.tif2991K

Fig. S5.In vivo analysis of homotypic fusion between Coxiella vacuoles in cells overexpressing EGFP-Vamp8.

A. HeLa cells transiently overexpressing EGFP-Vamp8 were infected with C. burnetii (moi = 10). After 24 h the cells were mounted into an in vivo camera and analysed by confocal microscopy for 42 min.

B. Inset showing the final stage of the fusion. C. Insets of the experiment described in A, showing the sequences of the movie (every 5 min). Red arrows indicate the CRVs, white arrows point to the fusion site.

cmi12087-sup-0006-si.avi2190K

Movie S1. Homotypic fusion between C. burnetii vacuoles in cells overexpressing EGFP-Vamp7 overexpression. HeLa cells transiently overexpressing EGFP-Vamp7 wt were infected with C. burnetii (moi = 10). After 24 h the cells were mounted into an in vivo camera and analysed by confocal microscopy for 70 min. The arrow indicates the marked enrichment of Vamp7 at the docking and fusion site.

cmi12087-sup-0007-si.avi1383K

Movie S2. Homotypic fusion between C. burnetii vacuoles in cell overexpressing EGFP-Vamp8 overexpression. HeLa cells transiently overexpressing EGFP-Vamp8 were infected with C. burnetii (moi = 10). After 24 h the cells were mounted into an in vivo camera and they were analysed by confocal microscopy for 42 min. The arrow points to the fusion site. No accumulation of Vamp8 is observed.

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