Fatty acid composition analysis confirms that <i>Mtb</i> incorporates host TAG-derived fatty acids directly into TAG.

<p><b>A</b> and <b>B</b>, Macrophage TAG labeled with [¹⁴C]oleate is utilized by <i>Mtb</i> for TAG accumulation. <b>A</b>, AgNO₃-TLC of methyl esters of fatty acids (FAMEs) prepared from TAG of <i>Mtb</i>-infected macrophages (lane 1, from left) and TAG from <i>Mtb</i> recovered from such macrophages (lane 2). <b>B</b>, Reversed-phase TLC analysis of FAMEs prepared from macrophage TAG (lane 1) and <i>Mtb</i> TAG (lane 2). Autoradiograms of the TLC plates with authentic ¹⁴C-labeled C16:0, C18:0, C18:1 and C20:4 FAMEs are shown. The AgNO₃-TLC and reversed-phase TLC show that ¹⁴C-oleic acid is incorporated into THPM TAG which is utilized to accumulate [¹⁴C]oleate-labeled TAG inside <i>Mtb</i>. <b>C</b>, FAMEs prepared from THPM and <i>Mtb</i> TAG analyzed using a Varian CP-TAP CB capillary column attached to a Varian CP-3900 gas chromatograph under a temperature control program. <i>Mtb</i> TAG FAMEs are identical to THPM TAG FAMEs except for very long-chain derivatives seen only in the TAG from the pathogen.</p

Dormancy and lipid metabolism genes are upregulated in <i>Mtb</i> recovered from lipid-loaded macrophages.

<p>TaqMan real-time PCR was used to measure the transcript levels of <i>Mtb</i> genes reported to be highly upregulated in a meta-analysis of <i>Mtb</i> microarray data from experimental models that mimicked dormancy. <i>Mtb</i> was recovered from lipid-loaded host cells at 72 h after incubation under hypoxia (1% O₂; 5% CO₂). Total RNA was reverse transcribed, the resulting cDNA was pre-amplified by multiplex-PCR with multiple <i>Mtb</i> gene-specific primers and the pre-amplified product was used in quantitative (q) PCR. Data was analyzed by ‘GenEx’ qPCR data analysis software (MultiD Analyses AB, Sweden) and gene transcript level was expressed as fold change in log2 scale relative to the sample from 18 h time point following normalization with 16S-rRNA as the reference gene. Average ± standard deviation from three replicates shown (n = 3); p<0.05, 18 h vs 72 h. <i>lip</i>, lipase, <i>tgs,</i> triacylglycerol synthase, <i>cut</i>, cutinase, <i>fcr</i>, fatty acyl-CoA reductase, <i>icl</i>, isocytrate lyase, <i>dosR</i>, dormancy response regulator, <i>hsp</i>, heat shock protein. The number prefixes are gene locus tag (Rv) numbers for respective <i>Mtb</i> genes.</p

<i>Mtb</i> replicates slowly inside hypoxic lipid-loaded macrophages. A

<p>and <b>B</b>, THPM incubated under hypoxia are viable hosts for <i>Mtb</i>. Uninfected (U) and infected (I) cells (MOI 0.1 or MOI 5.0) were incubated in either 1% O₂ or 21% O₂. THPM cell viabilities (<b>A</b>) and cell counts (<b>B</b>) were determined for the floating and adhered THPM populations. Data from triplicate measurements presented as average ± SD (n = 3). In <b>B</b>; *, statistically significant differences (p<0.005) between adhered vs floating populations; **, statistically insignificant differences (p>0.05) between 1% vs 21% incubations;. <b>C</b>, <i>Mtb</i> replication inside hypoxic THPM is severely curtailed in contrast to normoxic THPM. THPM were infected at an MOI of 0.1 and incubated under 1% O₂ or 21% O₂. At 0, 3, 5-days, <i>Mtb</i> CFUs were determined by agar plating. <i>Mtb</i> CFUs were normalized to THPM numbers. Data from triplicate measurements presented as average ± SD (n = 3); *, statistically significant differences (p<0.05) between 1% O₂ vs. 21% O₂ incubations.</p

<i>Mtb</i> inside lipid-loaded macrophages imports host fatty acids for storage as TAG.

<p>THPM were double isotope labeled with triolein [glycerol-1,2,3-³H, carboxyl-1-¹⁴C] (<b>A</b>) or oleic acid [9,10-³H, 1-¹⁴C] (<b>B</b>) for 24 h in 1% O₂, 5% CO₂ prior to <i>Mtb</i> infection at an MOI of 5. All 3500 x g pellets were detergent-washed and lipase-treated prior to lipid extraction as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002093#s4" target="_blank">Methods</a>. Total lipid extracts of dual isotope-labeled THPM and <i>Mtb</i> recovered from THPM were analyzed at 72 h post-infection. Lipids were resolved on silica TLC using hexane: diethyl ether: formic acid, 40∶10∶1 by volume, as solvent system and autoradiograms are shown. 3500 x g pellets of uninfected host cell lysates show no cross-contamination with host TAG (Lanes “UI” in <b>A, B</b>). Arrows indicate the relative positions of authentic internal lipid standards. UI, Uninfected background control, WE, wax esters; TAG, triacylglycerol; FA, fatty acids; PL, polar lipids.</p

Addition of anti-TNF α monoclonal antibody caused reactivation of dormant <i>Mtb</i>.

<p>(<b>A</b>) Auramine-O and Nile red staining of WT <i>Mtb</i> from IgG and anti-TNF α antibody treated granulomas. (<b>B</b>) Alamar Blue readouts at different time intervals when WT <i>Mtb</i> cells from granulomas treated with anti-TNF-α antibody or control IgG, were incubated with Alamar blue dye. (data of one representative data set has been displayed.) (<b>C</b>) WT <i>Mtb</i> cells from granulomas treated with anti-TNF-α antibody had a lower antibiotic resistance as compared to WT <i>Mtb</i> cells from control IgG treated granulomas. (<b>D</b>) <i>Mtb</i> cells from granulomas treated with anti-TNF α antibody catabolized fatty acids at a higher rate than <i>Mtb</i> from control IgG treated granuloma. P values were calculated using students t test. p<0.05 for metabolism of oleic and palmitic acid for <i>Mtb</i> from anti-TNF-αantibody treated granuloma compared to <i>Mtb</i> from IgG treated granuloma. (E) Infection of hPBMCs resulted in Apoptosis of these cells. Data are presented as mean +/− SEM from 3 experiments. P values were calculated using students t test. * p<0.05.</p

Deletion of <i>lipY</i> compromised the ability of <i>Mtb</i> to reactivate upon treatment with anti-TNFα mAb.

<p>(<b>A</b>) Auramine-O and Nile red staining of WT and <i>Δ-lip Y</i> mutant and <i>Δ-lip Y complemented strain (Δ-lip Y C+)</i> population in <i>in vitro</i> grown granuloma. (<b>B</b>) Alamar Blue readouts at different time intervals when WT and <i>Δ-lip Y, and Δ-lip Y C+ Mtb cells</i> from granulomas treated with anti-TNF-α antibody or control IgG, were incubated with Alamar blue dye. (data of one representative data set has been displayed.) (<b>C</b>) <i>Δ-lip Y</i> mutant from granulomas treated with IgG or anti-TNF-α antibody had a higher antibiotic resistance as compared to WT <i>Mtb</i> cells from anti-TNF-α antibody treated granulomas. Data are presented as mean +/− SEM from 3 experiments. P values were calculated using students t test. *p<0.05.</p

<i>Mtb</i> infection resulted in a decrease in macrophage and CD4+ T cell number.

<p>Granuloma samples were harvested and host cells were analyzed for expression of cellular markers by flow cytometry as described in methods. Graphs depict 1 wk and 2 wk post-infection profiles of uninfected and infected granuloma samples for (<b>A</b>) CD4+3+ T cells, (<b>B</b>) CD8+3+ T cells, (<b>C</b>) CD19+ B cells, (<b>D</b>) CD4+25+ T cells, (<b>E</b>) CD14+11c+ macrophages. Data are represented as mean +/− SEM from 3 experiments.* p<0.05 for CD4+25+ cells at 2 wk time point for 1∶0.1 MOI-infected group compared to the uninfected group.</p

Infection of human PBMC with <i>Mycobacterium tuberculosis</i> resulted in the formation of microscopic granulomas.

<p>(<b>A</b>) Infected PBMCs, (<b>B</b>) uninfected PBMCs, (<b>C</b>) H & E staining showing micro granulomas, (<b>D</b>) H & E staining showing multinucleated giant cells. (<b>E</b>) Fluorescent staining of granulomas sections with DAPI (nuclear stain), CD68 (macrophage marker-shown in red) and CD3 (T cells-shown in green) monoclonal antibodies.</p

<i>Mtb</i> within the granuloma goes into a dormant state.

<p>(<b>A</b>) Auramine-O and Nile red staining of granuloma sections shows Nile Red positive <i>Mtb</i> cells within the granuloma (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053657#pone-0053657-g004" target="_blank">figure 4A</a> lower left panel, insert in 4A shows lipid bodies). The number of Auramine-O and Nile red positive <i>Mtb</i> cells were counted from multiple fields, and percentage of Auramine-O (green) and Nile red (red) positive <i>Mtb</i> cells are presented graphically (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053657#pone-0053657-g004" target="_blank">Figure 4A</a> lower right panel) (<b>B</b>) <i>Mtb</i> from 8 day granuloma was resistant to Rif when compared to 0 day <i>in vitro</i> grown <i>Mtb</i>. Data are represented as mean +/− SEM from 3 experiments. P values were calculated using students t test. * p<0.05 for Rif resistance of <i>Mtb</i> from 8day granuloma compared to <i>Mtb</i> from 0day in vitro grown <i>Mtb</i>.</p

Gene expression profile of <i>Mtb</i> cells from <i>in vitro</i> granuloma treated with IgG anti-TNFα-mAb.

<p>Relative gene expression values (fold changes of day 0) in IgG and anti-TNFα mAb treated Mtb cells are represented for a selected set of genes that are known to be involved in dormancy and resuscitation conditions. Real-time Taqman RT-PCR measurement was performed to measure relative abundance of transcripts. Relative quantitation method (ddCt) was used to determine the fold change in transcripts level. Gene transcript level is expressed as fold change in log2 scale relative to the sample from in vitro grown starter culture used for infection of PBMC. Samples of starter culture (day 0) was used as calibrator and 16S rRNA gene was used as the endogenous control to normalize the expression values. tgs1, triacylglycerol synthase1; lipY, lipase Y; icl, isocitrate lyase; hspX, heat-shock protein X; dosR, dormancy response regulator; gltA1, citrate synthase 1; citA, citrate synthase II; rpf, resuscitation promoting factor (A, B, and C); rpo, RNA polymerase (A and B); atp, ATP synthase (A and B subunits); nuo, NADH dehydrogenase (A, B and E subunits).</p