Lipidomic Analysis Links Mycobactin Synthase K to Iron Uptake and Virulence in <i>M</i>. <i>tuberculosis</i>

<div><p>The prolonged survival of <i>Mycobacterium tuberculosis</i> (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual <i>mbt</i> genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth <i>in vitro</i> and <i>in vivo</i>. The deletion mutant, Δ<i>mbtN</i>, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of <i>mbtK</i> ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The <i>mbtK</i> mutant showed markedly reduced iron scavenging and growth <i>in vitro</i>. Further, Δ<i>mbtK</i> was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.</p></div

<i>mbtK</i> is required for growth during iron-starvation and early virulence <i>in vivo</i>.

<p>(<b>A</b>) Liquid M. tb cultures grown in iron-depleted medium, supplemented or not with 50 μM ferric chloride. (<b>B</b>) Representative plates from three replicates of the strains shown in (A) grown for 3 weeks on iron-depleted plates supplemented or not with 50 μM ferric chloride. (<b>C</b>) Δ<i>mbtK</i> and complemented Δ<i>mbtK</i>, marked chromosomally with unique identifiers (q-tags), were mixed 50:50 and used to infect fifteen C57/B6 mice at ~1,000 CFU via aerosol. Five mice were sacrificed at each time point. Lung homogenates were plated for CFU, colonies were counted and collected from plates to prepare genomic DNA. Average total recovered CFU were 1,131, 68,355 and 655,650 at 24 hours, 1 week and 6 weeks, respectively. Quantitative PCR for the q-tag specific to each strain was performed in duplicate, resulting in chromosomal equivalents (CEQ) of each strain to the total CEQ recovered per lung [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004792#ppat.1004792.ref037" target="_blank">37</a>]. Log ratios were evaluated by unpaired T-tests.</p

<i>mbtK</i> deletion decreases phospholipid abundance during iron starvation.

<p>Mean intensities of representative lipids from 5 glycerophospholipid classes and mycobactin control were normalized to their value in triplicate iron-depleted wild type cultures grown and analyzed in triplicate. Dashed line indicates the ion intensity in iron-depleted wild type. Statistical comparisons are Student’s t-tests of ion intensities in iron-supplemented wild type and iron-depleted Δ<i>mbtK</i>. MB = mycobactin, PE = phosphotidylethanolamine, PI = phosphotidylinositol, CL = cardiolipin, PG = phosphatidylglycerol, TAG = triacylglyceride, FA = fatty acid.</p

<i>mbtK</i> deletion depletes phosphatidylinositol during iron starvation.

<p><b>(A)</b> Phosphatidylinositol C35:0, neutral mass 852.5728, was detected in the positive mode (<b>B</b>) as [M+H]⁺ (<i>m/z</i> 853.5802) from triplicate total lipid extracts that were normalized for mass. Chromatogram is representative of triplicate runs. (<b>C</b>) Collision of phosphatidylinositol C35:0 in the negative mode as the [M-H]^- ion, <i>m/z</i> 851.5655, showed the expected fragments confirming its structure.</p

Δ<i>mbtN</i> lacks unsaturated mycobactin.

<p>(<b>A</b>) Acetone precipitation of lipids from three replicate cultures grown on iron-depleted agar were detected by three independent runs on reversed phase HPLC-MS to generate aligned datasets in XCMS software. Features increased or decreased by ≥ two-fold fall outside the dashed lines. Highlighted ions match the highlighted calculated <i>m/z</i> values in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004792#ppat.1004792.g001" target="_blank">Fig. 1C</a>, corresponding to: saturated mycobactins (white diamonds; <i>m/z</i> 911.4739, <i>m/z</i> 925.4883 and <i>m/z</i> 939.5008), unsaturated mycobactins (black diamonds; <i>m/z</i> 909.4564, <i>m/z</i> 923.4726 and <i>m/z</i> 937.4869), saturated monodeoxymycobactins (white circles; <i>m/z</i> 842.5663, <i>m/z</i> 856.5809, <i>m/z</i> 856.5828 and <i>m/z</i> 881.4590), unsaturated monodeoxymycobactins (black circles; <i>m/z</i> 865.4293, <i>m/z</i> 893.4444, and <i>m/z</i> 907.4781), saturated dideoxymycobactins (white squares; <i>m/z</i> 826.5531 and <i>m/z</i> 840.5852), and unsaturated dideoxymycobactins (black squares; <i>m/z m/z</i> 824.5594 and <i>m/z</i> 838.5714). All molecules detected as [M+Fe-2H]⁺, except <i>m/z</i> 856.5794 and dideoxymycobactins, detected as [M+H]⁺. (<b>B</b>) Representative ion chromatograms from three replicates from wild type (grey), Δ<i>mbtN</i> (purple) and Δ<i>mbtN</i> complement (dashed red) corresponding to unsaturated mycobactin <i>m/z</i> 923.4701, monodeoxymycobactin <i>m/z</i> 907.4754 and dideoxymycobactin <i>m/z</i> 838.5688; saturated mycobactin <i>m/z</i> 925.4858, monodeoxymycobactin <i>m/z</i> 909.4909 and dideoxymycobactin <i>m/z</i> 840.5845 (not detectable above background in wild type). (<b>C</b>) Collisional MS of unsaturated mycobactin (<i>m/z</i> 923.4701) from wildtype M. tb and saturated mycobactin (<i>m/z</i> 925.4858) from Δ<i>mbtN</i>. The constant presence of <i>m/z</i> 355, corresponding to the peptide-polyketide backbone, accompanied by a characteristic two-mass unit difference in unsaturated and saturated fragments separating the phenoloxazoline and acyl chain from the cobactin (<i>m/z</i> 711 and 713), isolate the mass difference to the fatty acyl unit.</p

Mycobactin synthases synthesize M. tb siderophores mycobactin and carboxymycobactin.

<p>(<b>A</b>) The <i>mbt-2</i> locus encodes mycobactin synthases that generate and modify the lipidic portion of mycobactin and carboxymycobactin. (<b>B</b>) M. tb mutants in <i>mbtN</i> and <i>mbtK</i> were generated by homologous recombination between pJM1::Δ<i>mbtN</i> or pJM1::Δ<i>mbtK</i> and flanking regions. Dots, <i>hyg</i> hygromycin resistance cassette and <i>sacB Bacillus subtilis</i> levansucrase gene. (<b>C</b>) The structures mycobactin T, monodeoxymycobactin, dideoxymycobactin and carboxymycobactin T are shown with the potential unsaturation indicated by a dashed line. In addition to the depicted core structures, known variations in the fatty acyl chain, listed below as <i>m/z</i> values of [M+H]⁺ or [M+Fe-2H]⁺ adducts, were calculated and used to search the lipidomics datasets. Monodeoxymycobactin forms both [M+H]⁺ and [M+Fe-2H]⁺ adducts. Apo-mycobactin was not detected. For carboxymycobactin, R = H or CH₃. Detected ions, highlighted in red, are highlighted in scatter plots in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004792#ppat.1004792.g002" target="_blank">Fig. 2A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004792#ppat.1004792.g003" target="_blank">3A</a>.</p

<i>mbtK</i> is required for mycobactin biosynthesis.

<p>(<b>A</b>) Three biological replicates of wild type M. tb or Δ<i>mbtK</i> were grown on iron-depleted agar and represented as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004792#ppat.1004792.g002" target="_blank">Fig. 2A</a>. Ions highlighted correspond to saturated mycobactins (white diamonds; <i>m/z</i> 911.4753, <i>m/z</i> 925.4799 and <i>m/z</i> 939.4877), unsaturated mycobactins (black diamonds; <i>m/z</i> 909.4624, <i>m/z</i> 923.4782 and <i>m/z</i> 937.4869), saturated monodeoxymycobactin (white circle; <i>m/z</i> 856.5665), unsaturated monodeoxymycobactins (black circles; <i>m/z</i> 840.5532, <i>m/z</i> 865.4293, <i>m/z</i> 893.4444, <i>m/z</i> 893.4641 and <i>m/z</i> 907.4783), and unsaturated dideoxymycobactins (black squares; <i>m/z</i> 810.5370, <i>m/z</i> 824.5598 and <i>m/z</i> 838.5714). (<b>B</b>) Representative ion chromatograms from three replicates corresponding to unsaturated mycobactin <i>m/z</i> 923.4701, monodeoxymycobactin <i>m/z</i> 907.4754, and dideoxymycobactin <i>m/z</i> 838.5688; saturated mycobactin <i>m/z</i> 925.4858 monodeoxymycobactin <i>m/z</i> 909.4909, and dideoxymycobactin <i>m/z</i> 840.5845 (not detectable above background).</p