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

Abstract

pathogenesis ( 4-6 ). The abundance and biological importance of the Mtb lipids has resulted in extensive and elegant studies to elucidate their structures and functions ( 1-3 ). In many cases, the lipids of Mtb are unique to this pathogen or shared only with other members of this genus.Earlier studies demonstrate variability in lipid profi les among different strains of Mtb ( 7-11 ) and that minor variations in the structure of individual lipids can occur with changes in the growth environment (12)(13)(14)(15)(16)(17)(18)(19)(20). However, targeted and nontargeted assays that monitor changes in Mtb lipid profi les are generally performed by traditional TLC-based methods ( 21 ), and global lipidomics analyses in Mtb have been restricted due to limits in the technology to detect and rapidly identify a large number of lipids in a single experiment. Two-dimensional NMR was recently applied to examine global mycobacterial lipid profi les, and this approach allowed for the identifi cation of key lipid differences in 13 C-enriched cellular extracts ( 22 ). Although this approach easily detects changes in lipid patterns, it is limited by the complexity of the NMR spectra and the overlapping chemical properties of many lipids. Alternatively, MS-based lipidomic strategies allowing simultaneous detection, identifi cation, and quantifi cation of structurally diverse lipid components of Mtb also were evaluated. Leavell and Leary ( 23 ) developed an algorithm to analyze high-resolution Fourier transform-ion cyclotron 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 profi ling. This methodology is based on efficient chromatographic separation and automated ion identifi cation 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

Detailed Structural and Quantitative Analysis Reveals the Spatial Organization of the Cell Walls of in Vivo Grown Mycobacterium leprae and in Vitro Grown Mycobacterium tuberculosis

“…The fragmentor voltage was set to 120 V. The mass spectrum was acquired from m/z 250 to 3200 Da with a frequency of 1 scan/s. MS data were analyzed using Mass Hunter software (Agilent) and a custom data base of M. tuberculosis lipids (40).…”

Lipidomic discovery of deoxysiderophores reveals a revised mycobactin biosynthesis pathway in Mycobacterium tuberculosis

“…S1B). Other applications have sought to catalog known mycobacterial lipids at the outset of the experiment and globally track many named molecules (untargeted lipidomics) (22,23). Our targeted lipidomics approach ranks unknown lipids according to a biological criterion (lipids consistent with mycobactin metabolites), and then solves their structures through collisional MS.…”

Broadening Our View About the Role of Mycobacterium tuberculosis Cell Envelope Components During Infection: A Battle for Survival

“…Efforts in improving purification techniques are also allowing us to be able to purify M.tb directly from infected tissues. The use of novel state-of-the art mass spectrometry techniques such as LC/MS/MS (Sartain et al, 2006(Sartain et al, , 2011, ESI/MS and MALDI-Tissue Imaging (Prideaux et al, 2011) in drug discovery will allow us to obtain new information about the cell wall composition from a single M.tb bacillus isolated from tissue and also to see how M.tb cell wall components, and other biomarkers and drugs are distributed within the infected tissue. These are few of many other novel biotechniques that are starting to be applied in the field of TB.…”