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cmi12217-sup-0001-figs1.tif10025KFig. S1. Actin and ArpC4 colocalize at the M. marinum compartment in Dictyostelium (related to Fig. 1). Dictyostelium expressing ArpC4-GFP was infected with M. marinum and fixed at 6 and 24 hpi. The fluorescent micrographs represent a confocal section of M. marinum (blue) that is surrounded by actin (red) and ArpC4-GFP (green) at 6 hpi (upper row) and 24 hpi (bottom row). Bars represent 2 μm.
cmi12217-sup-0002-figs2.tif4725K

Fig. S2. Deletion of wshA– leads to an absence of actin at the M. marinum compartment in Dictyostelium which can be restored by expression of GFP-WASH (related to Fig. 1F). Dictyostelium wild-type Ax2, wshA– and wshA– cells expressing GFP-WASH were infected with M. marinum. All strains were fixed at 6 and 24 hpi and stained for actin.

A. Quantitative microscopy using a confocal microscope showed that in wshA– cells actin is not associated with M. marinum vacuoles at 6 and 24 hpi (n = 3, mean ± SEM).

B. Complemented wshA– cells showed comparable levels of actin-association to the wild-type Ax2 (n = 3, mean ± SEM) as determined by confocal laser scanning microscopy.

cmi12217-sup-0003-figs3.tif3354KFig. S3. F-actin localizes to p80- but not VatA-positive M. marinum vacuoles (related to Fig. 3A). Dictyostelium wild-type Ax2 was infected with M. marinum and fixed at 6 hpi. Cells were stained for F-actin and either VatA or p80 respectively and the localization as well as colocalization of actin with either or VatA was counted using a laser scanning confocal microscope. F-actin associated M. marinum vacuoles were not found to colocalize with VatA at the same time (n = 3, mean ± SEM).
cmi12217-sup-0004-figs4.tif3824KFig. S4. VMC reduces the association of actin with M. marinum vacuoles (related to Fig. 2). Dictyostelium expressing VMC was infected with M. marinum and fixed at 6 and 24 hpi. Cells were stained for actin using phalloidin and the myc-tag to visualize VMC. Analysis by fluorescence microscopy showed that at 6 hpi VMC predominantly localizes to M. marinum vacuoles that are free of actin. The number of actin-positive compartments is reduced (actin) and only few vacuoles are positive for both markers (myc + actin). At later time points (24 hpi), only few vacuoles are associated with actin alone whereas most vacuoles were positive for VMC alone or VMC together with actin (myc + actin); (n = 3, mean ± SEM).
cmi12217-sup-0005-figs5.tif2168K

Fig. S5. Calmodulin, a marker for the contractile vacuole, does not localize to the M. marinum vacuole (related to Fig. 3). To assess in Dictyostelium whether VatM is delivered to the M. marinum vacuole by interactions with the contractile vacuole, calmodulin, a marker protein for the contractile vacuole, was localized in infected cells before and after treatment with LatA.

A. Confocal analysis of fixed Dictyostelium cells expressing VatM-GFP infected with M. marinum (blue, asterisk; 6 hpi) and treated with solvent alone. Immunofluorescence labelling was performed to localize calmodulin. VatM (red) but no calmodulin (green) is found around the mycobacterial vacuole. Both marker colocalize to the tubules of the contractile vacuole (indicated with white arrows).

B. Treatment with LatA (5 μM, 1 h) does not change the localization of calmodulin. Only VatM (red) but not calmodulin (green) localizes to the M. marinum (blue, asterisk; 6 hpi) vacuole. Bars represent 2 μm.

cmi12217-sup-0006-figs6.tif5247KFig. S6.M. marinum growth ‘in vitro’ is not affected by LatA (related to Fig. 4). To analyse whether LatA has an impact on M. marinum viability, we incubated bacteria with Dictyostelium growth medium (HL5c) at 25°C. Like during the infection the dish was incubated under static conditions. After 10, 24, 32 and 48 h an aliquot was collected and treated as described for cfu counting. Serial dilutions were plated on 7H11 plates and incubated at 32°C. The number of colonies was determined for each time point and normalized to the first value (10 h time point).
cmi12217-sup-0007-figs7.tif5825K

Fig. S7. In wshA– cells infection with M. marinum is not as efficient as in wild-type cells (related to Fig. 1E and F).

A. Dictyostelium wild-type Ax2 and wshA– cells were infected with M. marinum expressing GFP as described in the material and methods section. FACS analyses revealed an increase in green fluorescence over time (48 hpi). In wshA– cells the fluorescence was reduced in comparison to wild-type cells (Ax2).

B. Wild-type (Ax2), wshA– and wshA– expressing GFP-WASH were infected with M. marinum and fixed at 24 hpi. Cells were stained for actin using phalloidin and the number of ejectosomes per 100 infected cells was quantified using a widefield fluorescence microscope as described in the material and method section.

cmi12217-sup-0008-figs8.tif6649K

Fig. S8. WASH and F-actin colocalize with the M. tuberculosis vacuole (related to Fig. 5). RAW 264.7 macrophages were infected with M. tuberculosis (blue) and fixed at 2 and 4 hpi. The cells were stained for F-actin using phalloidin and WASH.

A. Quantification of association with F-actin at 2 and 4 hpi shows a significant lower number of actin-positive vacuoles at 4 hpi when compared with the earlier 2 hpi time point (n = 3, mean ± SEM, **P ≤ 0.01).

B. The micrograph shows a confocal section of an infected cell. While WASH and actin showed partial colocalization on structures in the cytosol (indicated by white arrows), no colocalization was observed around M. tuberculosis (blue). Bar represents 2 μm.

cmi12217-sup-0009-figs9.tif13028KFig. S9. Coronin-GFP localizes to actin- and p80- but not VatA-positive M. marinum vacuoles in Dictyostelium (related to Fig. 1G). Dictyostelium cells negative for coronin (corA–) or Ax2 wild type expressing Coronin-GFP were infected with M. marinum (blue) and fixed at 6 and 24 hpi. The cells were stained for p80, VatA and F-actin respectively. Immunofluorescence micrographs were collected with a laser scanning microscope. At 6 hpi coronin-GFP (green) localizes to compartments, that are decorated with p80 (red, first row) but not to VatA-positive vacuoles (red, second row). In contrast to wshA– cells, actin was associated with M. marinum compartments in corA– cells (6 hpi). At 24 hpi coronin-GFP (green, third row) localizes to M. marinum vacuoles that are also decorated with actin (red). CorA– cells were infected with M. marinum and fixed at 6 hpi (bottom row). The cells were stained for actin using phalloidin. CorA– cells show actin association at the mycobacterial compartment. Bars represent 2 μm.
cmi12217-sup-0010-figs10.tif6290K

Fig. S10. Deletion of corA leads to a transient increase of V-ATPase association at the M. marinum compartment in Dictyostelium (related to Fig. 1G). Dictyostelium wild-type Ax2, corA– (HG1569) and wshA– cells were infected with M. marinum and fixed at 6 and 24 hpi. The cells were stained for immunofluorescence microscopy using antibodies against VatA and p80.

A. In corA– cells, association of M. marinum vacuoles with VatA is significantly increased at 6 hpi but not at 24 hpi (n = 3, mean ± SEM, ***P ≤ 0.001, n.s. = not significant). With respect to p80 (right graph) the corA– mutant shows no significant differences in the level of association compared with the wild-type Ax2.

B. In wshA– cells at 24 hpi the number of M. marinum vacuoles associated with VatA is significantly increased (n = 3, mean ± SEM, *P ≤ 0.05) but colocalization with p80 is reduced (n = 3, mean ± SEM, ***P ≤ 0.001), which is in contrast to our findings in Ax2 wild-type cells.

cmi12217-sup-0011-appendixs1.doc93KAppendix S1. Extended material and methods.

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