<i>Mtb</i> Δ<i>fcr</i> mutants show increased metabolic activity and induction of genes involved in energy generation and transcription under dormancy-inducing conditions.

<p><b>A, </b><i>Mtb</i> WT and <i>fcr</i> deficient cells subjected to combined MS condition for 0, 9 and 18 days were incubated with Alamar Blue dye for indicated periods of time and fluorescence was measured using a plate reader. <b>B,</b> Relative gene expression values (fold induction of day 0) of each gene for WT and Δ<i>fcr</i> mutants at 9 and 18 days under MS. Real-time Taqman RT-PCR measurement was performed to measure relative abundance of transcripts. Relative quantitation method (ddCt) was used with the 7900 HT real-time system and analysis was done using SDS v2.3 software of Applied Biosystems Inc. Samples of starter culture (day 0) were used as calibrator and <i>sigA</i> was used as the endogenous control to normalize expression values.</p

Wax Ester Synthesis is Required for <em>Mycobacterium tuberculosis</em> to Enter <em>In Vitro</em> Dormancy

<div><p><em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) is known to produce wax esters (WE) when subjected to stress. However, nothing is known about the enzymes involved in biosynthesis of WE and their role in mycobacterial dormancy. We report that two putative <em>Mtb</em> fatty acyl-CoA reductase genes (<em>fcr</em>) expressed in <em>E. coli</em> display catalytic reduction of fatty acyl-CoA to fatty aldehyde and fatty alcohol. Both enzymes (FCR1/Rv3391) and FCR2/Rv1543) showed a requirement for NADPH as the reductant, a preference for oleoyl-CoA over saturated fatty acyl-CoA and were inhibited by thiol-directed reagents. We generated <em>Mtb</em> gene-knockout mutants for each reductase. Metabolic incorporation of¹⁴C-oleate into fatty alcohols and WE was severely diminished in the mutants under dormancy-inducing stress conditions that are thought to be encountered by the pathogen in the host. The fatty acyl-CoA reductase activity in cell lysates of the mutants under nitric oxide stress was significantly reduced when compared with the wild type. Complementation restored the lost activity completely in the Δ<em>fcr1</em> mutant and partially in the Δ<em>fcr2</em> mutant. WE synthesis was inhibited in both Δ<em>fcr</em> mutants. The Δ<em>fcr</em> mutants exhibited faster growth rates, an increased uptake of ¹⁴C-glycerol suggesting increased permeability of the cell wall, increased metabolic activity levels and impaired phenotypic antibiotic tolerance under dormancy-inducing combined multiple stress conditions. Complementation of the mutants did not restore the development of antibiotic tolerance to wild-type levels. Transcript analysis of Δ<em>fcr</em> mutants showed upregulation of genes involved in energy generation and transcription, indicating the inability of the mutants to become dormant. Our results indicate that the <em>fcr1</em> and <em>fcr2</em> gene products are involved in WE synthesis under <em>in vitro</em> dormancy-inducing conditions and that WE play a critical role in reaching a dormant state. Drugs targeted against the <em>Mtb</em> reductases may inhibit its ability to go into dormancy and therefore increase susceptibility of <em>Mtb</em> to currently used antibiotics thereby enhancing clearance of the pathogen from patients.</p> </div

Acyl-CoA reductase activity of FCR1 and FCR2-expressing <i>E. coli</i> lysates.

<p>Acyl-CoA reductase activity was measured with 70 µg protein in a final volume of 250 µl of 0.1 M phosphate buffer for 45 min (FCR1) and 30 min (FCR2). The dependence of enzymatic activity on pH (A, E), NADPH concentration (B, F), oleoyl-CoA concentration (C, G) and inhibition of alcohol and aldehyde formation by thiol-directed reagents (D, H) was determined to confirm the authenticity of the observed acyl-CoA reductase activity in lysates. Values are average ± SD from three independent experiments.</p

<i>E. coli</i> lysates express enzymatically active FCR1 and FCR2 proteins with negligible background activity.

<p><b>A,</b> Cell lysates of <i>E. coli</i> expressing the reductases were analyzed by SDS-PAGE followed by coomassie staining<b>.</b> Lane 1: Molecular weight (MW) markers, Lane 2: Untransformed <i>E. coli</i> (BL21) lysate, Lane 3: FCR1-expressing <i>E. coli</i> lysate, Lane 4: FCR2-expressing <i>E. coli</i> lysate. Arrows indicate the approximately 74 kDa FCR1 protein and 40 kDa FCR2 protein. <b>B,</b> Untransformed <i>E. coli</i> BL21 lysate does not show significant acyl-CoA reductase activity. Autoradiogram of TLC analysis of reaction products shown. BL21, lysate of untransformed <i>E. coli</i> BL21 host cells; Radiolabeled palmitoyl-CoA (C16∶0), stearoyl-CoA (C18∶0) or oleoyl-CoA (C18∶1) were provided as substrates. Arrows indicate positions of authentic lipid standards. CHO, fatty aldehydes; FA, fatty acids; FA-OH, fatty alcohols; PL, polar lipids.</p

Southern blot analysis of <i>Mtb</i> Δ<i>fcr</i>1 and Δ<i>fcr</i>2 mutants.

<p><b>A,</b> Schematic depiction shows the genomic locations of the primers and probes used in the construction and confirmation of <i>fcr</i> deletion mutants. The sequences of the primers are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051641#pone.0051641.s001" target="_blank">Table S1</a>. <b>B,</b> Genomic DNA from WT <i>Mtb</i> and Δ<i>fcr</i>1 mutant was digested with <i>Eco</i>RI and hybridized with the 3′-flank of the Δ<i>fcr</i>1 construct as probe (P1). The WT <i>fcr1</i> contains two <i>Eco</i>RI sites in the deleted part of the gene, the last one being only 48 bp upstream of the 3′ flank region of the construct. When this 3′ flank sequence was used as the probe, it hybridized to a 4930 bp fragment of the <i>Eco</i>RI digested genomic DNA (lane WT). When the hyg cassette replaced the native gene sequence, its <i>Eco</i>RI site was situated 1047 bp upstream of the 3′ flank sequence which resulted in a shift of the WT band to 5929 bp (lane fcr1). <b>C,</b> Genomic DNA from WT <i>Mtb</i> and Δ<i>fcr</i>2 mutant was digested with <i>Pst</i>I and hybridized with the 5′-flank of the Δ<i>fcr</i>2 construct as the probe (P3). Wild-type genomic DNA digested with <i>Pst</i>I and probed with the 5′ flank of the disruption construct yielded a hybridization fragment of 3292 bp (lane WT). In contrast <i>Pst</i>I digested DNA from the mutant strain showed a smaller band of 1741 bp due to the presence of a <i>Pst</i>I site in the 5′ region of the hyg cassette (lane fcr2).</p

<i>Mtb</i> Δ<i>fcr</i> mutants are severely impaired in developing phenotypic antibiotic tolerance under dormancy-inducing MS conditions.

<p><i>Mtb-</i>WT, its <i>fcr</i> gene-knockout mutants (Δ<i>fcr1</i> & Δ<i>fcr2</i>) and their complemented strains (C-<i>fcr1</i> & C-fcr2) were subjected to MS for up to 18 days and antibiotic tolerance of the cultures against rifampicin (5 µg/ml) and isoniazid (0.8 µg/ml) were measured as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051641#pone.0051641-Deb1" target="_blank">[11]</a>. Average (±SD) from three different experiments (n = 3) is shown. MS, multiple-stress.</p

<i>Mtb</i> Δ<i>fcr</i> mutants show increased nutrient uptake under dormancy-inducing conditions <i>in vitro</i>.

<p><i>Mtb</i> WT and <i>fcr</i>-deficient mutants and complemented mutants subjected to MS conditions were incubated with [¹⁴C]glycerol for different time periods. Washed cell pellets were used to measure the radioactivity inside cells as described in Methods. At 0-day, <i>Mtb</i> cells (in log-phase) displayed very low uptake of radiolabeled glycerol (<b>A</b>). At 9-days (<b>B</b>) and 18-days (<b>C</b>) under MS, in contrast to WT, <i>fcr</i>-deficient mutants and complemented mutants displayed highly elevated levels of [¹⁴C]glycerol uptake. WT, wild-type; <i>d-fcr1</i>, <i>fcr1</i>-deletion mutant; <i>C-fcr1</i>, complemented <i>fcr1</i>-deletion mutant; <i>d-fcr2</i>, <i>fcr2</i>-deletion mutant; <i>C-fcr2</i>, complemented <i>fcr2</i>-deletion mutant. DPM, disintegrations per minute.</p

Identification of <i>Mtb</i> fatty acyl-CoA reductases.

<p><b>A,</b> Pathway of wax ester biosynthesis. Fatty acyl-CoA reductase (FCR) catalyzes the reduction of fatty acid to fatty alcohol which is condensed with fatty acyl-CoA by wax synthase (WS) to generate wax ester. R, alkyl chain. <b>B,</b> Amino acid sequence alignment of FCR1 (Rv3391) and FCR2 (Rv1543) with the acyl-CoA reductases ACR1 of <i>Acinetobacter,</i> MAQU_2507 and MAQU_2220 of <i>Marinobacter</i>.</p

<i>Mtb</i> Δ<i>fcr</i> mutants are impaired in WE biosynthesis under combined MS.

<p><b>A,</b> Diminished incorporation of ¹⁴C-oleate into WE in <i>Mtb</i> Δ<i>fcr</i> mutants under combined MS treatment. <i>Mtb</i> cultures exposed to combined MS for 9 days were metabolically labeled with ¹⁴C-oleic acid for 4 h. Total lipids (equal proportions across samples) were resolved on silica-TLC and autoradiograms from a typical experiment is shown. WT, wild type <i>Mtb</i>; d-fcr1, <i>fcr1</i>-deletion mutant; C-fcr1, complemented <i>fcr1</i> mutant; d-fcr2, <i>fcr2</i>-deletion mutant; C-fcr2, complemented <i>fcr2</i>-deletion mutant. Arrows indicate relative positions of authentic lipid standards. WE, wax esters; TG, triacylglycerols; FA, fatty acids; FA-OH, fatty alcohols, PL, polar lipids. <b>B.</b> Loss of <i>fcr1</i> or <i>fcr2</i> results in diminished wax ester biosynthesis under combined MS. Complementation restores WE biosynthesis in <i>fcr1</i>-deletion mutant only. Radioactivity from ¹⁴C-oleate incorporated into fatty alcohol and WE after 9 days under combined MS was determined and normalized as percent of total radioactivity in the respective lipid extract. Values are average ± SD.</p

WE and fatty alcohol synthesis are impaired in <i>Mtb</i> Δ<i>fcr</i> mutants under dormancy-inducing nitric oxide treatment.

<p><b>A,</b> Fatty alcohol and WE formation are diminished in lysates of <i>Mtb fcr1</i> and <i>fcr2</i> deletion mutants subjected to NO-stress. Complementation restores lost activity completely in <i>d-fcr1</i> and partially in <i>d-fcr2</i> lysates. <i>Mtb</i> cells were exposed to nitric oxide and lysates were assayed for acyl-CoA reductase activity using ¹⁴C-oleoyl-CoA as substrate as described in Methods. Autoradiogram of TLC plate from a typical experiment is shown. WT, wild-type; <i>d-fcr1</i>, <i>fcr1</i>-deletion mutant; <i>C-fcr1</i>, complemented <i>fcr1</i>-deletion mutant; <i>d-fcr2</i>, <i>fcr2</i>-deletion mutant; <i>C-fcr2</i>, complemented <i>fcr2</i>-deletion mutant. Arrows indicate relative positions of authentic lipid standards. WE, wax esters; TG, triacylglycerols; FA, fatty acids; FA-OH, fatty alcohols, PL, polar lipids. <b>B,</b> Radioactivity in WE and fatty alcohols was determined by scintillation counting and activities, normalized to total protein content in respective lysates, are shown. Values are average ± SD from duplicate experiments. <b>C,</b> NO-treated <i>Mtb</i> cells displayed severe decrease in non-radiolabeled WE accumulation. The non-radiolabeled total lipids, resolved on silica-TLC, were visualized by charring at 180°C and the TLC plate is shown. WE, wax esters; PL, polar lipids. <b>D,</b> the WE band in each lane of the TLC was quantitated by densitometry using an AlphaInnotech gel documentation system and the WE levels in the mutants, relative to WT (set at 100%), are represented. Values are average ± SD from duplicate experiments.</p