α⁺, α, and α’ mycolates.

<p>Positive-ion mode Q-Tof mass spectrometry of 1D TLC purified MAMEs derived from <i>S. rugosus</i> (high and middle bands) or <i>S. rotundus</i> (low band). The high migrating MAMEs with longer chain length, the middle migrating MAMEs with medium chain length, or the low migration MAMEs with shorter chain length were initially designated as α⁺, α, or α’ mycolate subclasses, respectively. The most intense molecular ions, observed here as ammonium adducts, present in each subclass are indicated in bold and italics.</p

Purification and analysis of <i>Segniliparus</i> mycolates.

<p>(A) Normal phase one-dimensional thin-layer chromatography (1D TLC) of saponified and esterified lipids from <i>M. tuberculosis (M. tb)</i>, <i>S. rugosus</i> and <i>S. rotundus</i>. From left to right: Lanes 1, 2, 3, and 7 were loaded with purified keto (1), methoxy (2), alpha (3), or mixed (7) MAMEs of <i>M. tuberculosi</i>s. Lanes 4, 5, 6 were loaded with methylated saponificates containing both fatty acyl methyl esters and MAMEs derived from <i>S. rotundus</i> (4), <i>S. rugosus</i> (5), or <i>M. tuberculosis</i> (<i>M. tb</i>, 6). (B) Positive-ion mode Q-Tof ESI-MS analysis of total MAMEs derived from <i>M. tuberculosis</i> (top panels), with insets showing mass values that deduce to chemical formulas having either 3 (α) or 4 (keto, methoxy) oxygen atoms and compared with MAMEs from <i>S. rotundus</i> and <i>S. rugosus</i> (middle and bottom panels).</p

Electron micrographs.

<p>Scanning electron micrographs of <i>S. rotundus</i> (A) and <i>S. rugosus</i> (B)<b>.</b> (C) Transmission electron micrograph of <i>S. rugosus</i>. The bands are labeled as indicated. MOM stands for mycolate outer membrane and CM stands for cytoplasmic membrane.</p

Collision induced dissociation mass spectrometry demonstrates alpha branch chain length.

<p>(A) Purified <i>S. rugosus</i> MAMEs found in the high migrating fraction on the TLC plate were dissolved in chloroform and methanol solution, loaded into a nanospray tip and analyzed by positive-ion mode electrospray ionization mass spectrometry with ion trapping. The ion at <i>m/z</i> 1368.5 [M+Na]⁺ corresponding to C92 mycolic acid methyl ester was subjected to CID-MS, yielding a fragment ion corresponding to a sodium adduct of the meroaldehyde chain (<i>m/z</i> 1013.9) which indicates a neutral loss of the α-chain-derived C22∶0 methyl ester. (B–C) Bacterial pellets of <i>S. rugosus</i> and <i>S. rotundus</i> were extracted with chloroform, diluted with methanol and analyzed by negative-ion mode electrospray ionization mass spectrometry. Both samples contain an ion at m/z 1330.3 ([M-H]⁻) corresponding to C92 mycolic acid, which was subjected to CID-MS analysis. The product ion 339 from <i>S. rugosus</i> (B) and ions 339 and 367 from <i>S. rotundus</i> (C) indicate the presence of C22∶0 and C24∶0 α-branch-derived fatty acids.</p

Stereoselective Synthesis of 1‑Tuberculosinyl Adenosine; a Virulence Factor of <i>Mycobacterium tuberculosis</i>

Despite its status as one of the world’s most prevalent and deadly bacterial pathogens, <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infection is not routinely diagnosed by rapid and highly reliable tests. A program to discover <i>Mtb</i>-specific biomarkers recently identified two natural compounds, 1-tuberculosinyl adenosine (1-TbAd) and <i>N</i>⁶-tuberculosinyl adenosine (<i>N</i>⁶-TbAd). Based on their association with virulence, the lack of similar compounds in nature, the presence of multiple stereocenters, and the need for abundant products to develop diagnostic tests, synthesis of these compounds was considered to be of high value but challenging. Here, a multigram-scale stereoselective synthesis of 1-TbAd and <i>N</i>⁶-TbAd is described. As a key-step, a chiral auxiliary-mediated Diels–Alder cycloaddition was developed, introducing the three stereocenters with a high exo endo ratio (10:1) and excellent enantioselectivity (>98% ee). This constitutes the first entry into the stereoselective synthesis of diterpenes with the halimane skeleton. Computational studies explain the observed stereochemical outcome

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> 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

<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

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