Lipidomic analyses of Mycobacterium tuberculosis based on accurate mass measurements and the novel "Mtb LipidDB"

Abstract

The cellular envelope of Mycobacterium tuberculosis is highly distinctive and harbors a wealth of unique lipids possessing diverse structural and biological properties. However, the ability to conduct global analyses on the full complement of M. tuberculosis lipids has been missing from the repertoire of tools applied to the study of this important pathogen. We have established methods to detect and identify lipids from all major M. tuberculosis lipid classes through LC/MS lipid profiling. This methodology is based on efficient chromatographic separation and automated ion identification through accurate mass determination and searching of a newly created database (Mtb LipidDB) that contains 2,512 lipid entities. We demonstrate the sensitive detection of molecules representing all known classes of M. tuberculosis lipids from a single crude extract. We also demonstrate the ability of this methodology to identify changes in lipid content in response to cellular growth phases. This work provides a customizable framework and resource to facilitate future studies on mycobacterial lipid biosynthesis and metabolism.

Fig. 1.

Organizational summary of the Mtb LipidDB and LIPID MAPS classification systems.

Fig. 2.

Identified MFs from positive-ion LC/MS analyses of an Mtb total lipid extract. Six hundred seventy-two MF ions were matched to lipid groups representing 25 lipid subclasses from the Mtb LipidDB. See supplementary material for lipid group abbreviations.

Fig. 3.

Identified MFss from negative-ion LC/MS analyses of an Mtb total lipid extract. Two hundred forty-eight MF ions were matched to lipid groups representing 24 lipid subclasses from the Mtb LipidDB. See supplementary material for lipid group abbreviations.

Fig. 4.

Ion response scatter plots for an Mtb total lipid extract dilution series. The observed ion volumes were selected from representative lipid group ions detected across a wide dynamic range. Values were plotted against the amount of lipid extract injected (bottom x axis) and the equivalent number of extracted Mtb cells (top x axis).

Fig. 5.

Comparison of TG profiles from Mtb growth phases. A: TG lipid group ion volumes with the same carbon number were combined and normalized to total TG ion volume (for individual TG lipid group profiles, see supplementary Fig. IV). Error bars indicate ±SD (n = 3). The inset shows relative TG abundance as percent total identified lipid group ion volumes. B: MS/MS spectra of the [M+NH₄]⁺ molecular ions for representative TG groups. The major logarithmic-phase TG (52:3) ion produced two major diacyl product ions at m/z 575.5010 and m/z 603.5308 that resulted from neutral losses of C18:1 and C16:1 FAs, respectively. The major stationary phase-specific TG (60:0) ion produced three major diacyl product ions at m/z 579.5345, m/z 691.6605, and m/z 719.6682, which indicated neutral losses of C26:0, C18:0, and C16:0 FAs, respectively.

Fig. 6.

Comparison of GP subclass unsaturated bond content from Mtb growth phases. Percent of each lipid subclass containing lipid groups with fatty acyl sum composition possessing zero to four unsaturated bonds. LOG, logarithmic phase; TRANS, transitionary phase; STAT, stationary phase.

Fig. 7.

Growth phase-dependent changes in DIM A molecular size. DIM A group ion volumes were normalized to total DIM A ion volumes. Error bars indicate ± SD (n = 3).