Journal: PLoS Pathogens

Article Title: Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages

doi: 10.1371/journal.ppat.1002093

Figure Lengend Snippet: THPM were allowed to metabolically incorporate the fluorescent fatty acid BODIPY 558/568 C 12 for 24 h and unincorporated label was removed by washing prior to infection with Mtb . A , Intact lipid-loaded macrophage viewed under TRITC filter showing lipid droplets (red) metabolically labeled with fluorescent fatty acid. Nucleus stained with DAPI and overlay shown. B , Differential interference contrast image of Mtb recovered from THPM and C , Image of the same Mtb cell viewed with TRITC filter showing fluorescent BODIPY fatty acid-labeled lipid droplets within the pathogen. D , Snapshot of the 3D image of an intact lipid-loaded macrophage. The 3D image is constructed from image stacks taken with the appropriate filter sets for each stain and overlayed. E , Snapshot of the 3D image (obtained as described for the THPM, with only the TRITC filter set) of an Mtb cell recovered from THPM 6 days after infection showing lipid droplets within the pathogen. F , TLC showing that fluorescent TAG is the predominant lipid in both THPM and in Mtb within THPM. THPM were metabolically labeled with BODIPY fatty acid for 24 h under 1% O 2 and then infected with Mtb and incubated for a further 36 h. A small aliquot of THPM lipids and most of the lipids from Mtb were applied to the TLC plate. After silica-TLC with hexane: diethyl ether: formic acid (40∶10∶1) as the solvent, the plate was imaged under UV illumination with Texas Red filter. G , Fluorescence maximum intensities in individual Mtb cells of the WT (red) and the tgs1 (blue) strains showing that lipid-droplet accumulation inside Mtb is impaired in the absence of tgs1 . Cells of both strains were recovered from THPM pre-labeled with the BODIPY 558/568 C 12 and infected at an MOI of 0.25. Measurements of fluorescence intensities were performed as described in . Values from a representative experiment shown (n = 3). wt, wild type Mtb ; tgs1 , Δ tgs1 mutant; AU, arbitrary units.

Article Snippet: Mtb cells, recovered from PBMC-derived macrophages (infected at MOI 0.1) at 3 and 5 days under hypoxia, were concentrated by centrifugation and stained with Auramine-O (TB Fluorescent Stain Kit M, Becton Dickinson, Sparks, MD) and with Nile Red (Invitrogen/Molecular Probes, Carlsbad, CA) following a previously published protocol and examined by confocal laser scanning microscopy (Leica TCS SP5; Leica Microsystems, Mannheim, Germany) with Z-stacking.

Techniques: Metabolic Labelling, Infection, Labeling, Staining, Construct, Incubation, Solvent, Fluorescence, Mutagenesis