A comparative lipidomics platform for chemotaxonomic analysis of Mycobacterium tuberculosis

Abstract

The lipidic envelope of Mycobacterium tuberculosis promotes virulence in many ways, so we developed a lipidomics platform for a broad survey of cell walls. Here we report two new databases (MycoMass, MycoMap), 30 lipid fine maps, and mass spectrometry datasets that comprise a static lipidome. Further, by rapidly regenerating lipidomic datasets during biological processes, comparative lipidomics provides statistically valid, organism-wide comparisons that broadly assess lipid changes during infection or among clinical strains of mycobacteria. Using stringent data filters, we tracked more than 5,000 molecular features in parallel with few or no false-positive molecular discoveries. The low error rates allowed chemotaxonomic analyses of mycobacteria, which describe the extent of chemical change in each strain and identified particular strain-specific molecules for use as biomarkers.

Figure 1. MycoMass database content

List of the lipids cataloged in the MycoMass database (Figure S1). This database follows the Lipid Maps organizational tree and uses lipid families’ names found in the mycobacterial literature in level 4. Phosphatidylinositol mannosides (PIM_x) contain 1 to 6 mannosyl residues (x) and sulfoglycolipids (AcxSGL) contain 2 to 4 fatty acyl chains (x). Alkylforms vary by the saturation and carbon length of acyl chains and/or by the length of carbon backbones.

Figure 2. Lipidomics platform

Lipid extracts (dark blue) enter a workflow involving a universal normal phase HPLC-MS system (black), software-assisted raw data extraction (lime green), computational comparative analysis (red), database and dataset annotation (purple), and molecular discovery through collisional mass spectrometry (light blue). This second generation system for comparative profiling emphasizes a single-step chromatography system, in contrast to a first generation method that uses fluid phase separation and multiple HPLC systems (Figure S2). (B) Extracted ion chromatograms of the overall features detected with high, intermediate and low intensity by analyzing M. tuberculosis H37Rv total lipids.

Figure 3. Validation of a universal normal-phase HPLC-MS detection

Structures (A) of diverse benchmarks lipids for HPLC-MS method optimization related to countercurrent gas (B), source voltage (C) as measured in biological replicates (D). Relationship of signal intensity derived from areas under the curves of ion chromatograms to input mass of total lipid (E). Data is representative of three or more experiments.

Figure 4. Mapping the lipidome of M. tuberculosis H37Rv

(A) HPLC-MS dataset of M. tuberculosis H37Rv of ~6,000 features, which are 3-D coordinates of linked m/z, retention time and intensity. One lead compound in each cluster was tentatively identified by automated annotation using MycoMass, confirmed by four analytical criteria and mapped to the chromatographic system in positive- (B) and negative-ion mode (C). Neutral formulas of the studied alkylforms and detected m/z of the respective [M+NH₄]⁺ or [M+H]⁺ adducts (B) and [M-H]⁻ forms (C) are indicated. Retention times of lipids typically vary by less than 5 seconds in one experiment, but vary up to 60 seconds among users with differing columns. Phosphatidylinositol mannosides (PIM_x) are listed according to the number x of mannosyl residues. 30 lipid families mapped in this way comprise the MycoMap. Features annotation and collisional MS are shown in Figure S4.

Figure 5. In vitro and in vivo fine mapping of M. tuberculosis H37Rv lipid families

Extracts of M. tb H37 Rv grown in vitro were subject to detection of PDIM, trehalose monomycolate (A-B) and 28 other lipids (Figure S5) illustrating the PDIM detected alkylforms (A) and retention time profiles (B) that match by color and confirmed by collisional mass spectrometry (C). (D) M. tuberculosis Erdman 2.5 grown in broth media or in one infected mouse were similarly analyzed for the PDIM A/A’ and B alkylforms with the indicated overall carbon number. Similar results were found in separate analysis of 3 mice (Figure S6).

Figure 6. Comparative chemotaxonomy

(A-D) Pairwise comparison of extractable lipids represented as volcano plots, showing in red the features meeting criteria for 2-fold change and significance (p < 0.05, corrected for multiple comparison) also indicated as a percentage of all features (n). M. tuberculosis H37Rv lipid extract from one (A) or two (B) liquid cultures were analyzed in triplicate and compared with M. tuberculosis Beijing HN878 (C) or M. smegmatis (D). Among features uniquely present in the W Beijing strain (C, inset and listed Figure S7) 38 (green) correspond to isotopes (M, M+1, M+2) and adducts (NH ⁺₄ or Na⁺) of a triglycosylated phenolic glycolipid (PGL) alkane series, as illustrated for two alkylforms of nominal masses of 1827 and 1841. (E) Collisional mass spectrometry of [M+NH₄]⁺ adduct of PGL confirmed structure composed of a phthiocerol core esterified by C27 and C30 mycocerosic acids (R1, R2). (F,G) Extracted ion chromatograms of a representative alkylform of the monoglycosylated (m/z 1553.442) or triglycosylated (m/z 1845.554) form of PGL for laboratory and patient isolates show sensitive detection that is not confounded by other lipids and separate detection of the two PGL glycoforms. The mass spectrum of triglycosylated PGLs is shown Figure S7B.