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Identification of specific metabolites in culture supernatant of Mycobacterium tuberculosis using metabolomics: exploration of potential biomarkers.

ABSTRACT: Although previous studies have reported the use of metabolomics for Mycobacterium species differentiation, little is known about the potential of extracellular metabolites of Mycobacterium tuberculosis (MTB) as specific biomarkers. Using an optimized ultrahigh performance liquid chromatography-electrospray ionization-quadruple time of flight-mass spectrometry (UHPLC-ESI-Q-TOF-MS) platform, we characterized the extracellular metabolomes of culture supernatant of nine MTB strains and nine non-tuberculous Mycobacterium (NTM) strains (four M. avium complex, one M. bovis Bacillus Calmette-Guérin (BCG), one M. chelonae, one M. fortuitum and two M. kansasii). Principal component analysis readily distinguished the metabolomes between MTB and NTM. Using multivariate and univariate analysis, 24 metabolites with significantly higher levels in MTB were identified. While seven metabolites were identified by tandem mass spectrometry (MS/MS), the other 17 metabolites were unidentified by MS/MS against database matching, suggesting that they may be potentially novel compounds. One metabolite was identified as dexpanthenol, the alcohol analog of pantothenic acid (vitamin B5), which was not known to be produced by bacteria previously. Four metabolites were identified as 1-tuberculosinyladenosine (1-TbAd), a product of the virulence-associated enzyme Rv3378c, and three previously undescribed derivatives of 1-TbAd. Two derivatives differ from 1-TbAd by the ribose group of the nucleoside while the other likely differs by the base. The remaining two metabolites were identified as a tetrapeptide, Val-His-Glu-His, and a monoacylglycerophosphoglycerol, phosphatidylglycerol (PG) (16?0/0?0), respectively. Further studies on the chemical structure and biosynthetic pathway of these MTB-specific metabolites would help understand their biological functions. Studies on clinical samples from tuberculosis patients are required to explore for their potential role as diagnostic biomarkers.

SUBMITTER: Lau SK

PROVIDER: S-EPMC4317673 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Identification of specific metabolites in culture supernatant of Mycobacterium tuberculosis using metabolomics: exploration of potential biomarkers.

Lau Susanna K P SK Lam Ching-Wan CW Curreem Shirly O T SO Lee Kim-Chung KC Lau Candy C Y CC Chow Wang-Ngai WN Ngan Antonio H Y AH To Kelvin K W KK Chan Jasper F W JF Hung Ivan F N IF Yam Wing-Cheong WC Yuen Kwok-Yung KY Woo Patrick C Y PC

Emerging microbes & infections 20150128 1

Although previous studies have reported the use of metabolomics for Mycobacterium species differentiation, little is known about the potential of extracellular metabolites of Mycobacterium tuberculosis (MTB) as specific biomarkers. Using an optimized ultrahigh performance liquid chromatography-electrospray ionization-quadruple time of flight-mass spectrometry (UHPLC-ESI-Q-TOF-MS) platform, we characterized the extracellular metabolomes of culture supernatant of nine MTB strains and nine non-tube ...[more]

PMID: 26038762

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Project description:Increasing evidence demonstrates that antigen-specific cellular and humoral immunity plays an indispensable role in protection against Mycobacterium tuberculosis infection. Antigen is a key element in the development of a successful diagnostic method and vaccine. However, few antigens are available, and a systemic study on M. tuberculosis ORFeome-based antigen screening is still lacking. In the current study, a genome-wide examination was conducted on high-throughput M. tuberculosis encoding proteins and novel antigens were identified via a comprehensive investigation of serological and antigen-specific cellular responses. The serological immunoglobulin G level of each protein was detected in pooled sera from 200 pulmonary tuberculosis patients by means of semi-quantitative Western blot. Of the 1,250 detected proteins, 29 were present at a higher level relative to the commercialized 38-kDa protein. Furthermore, the top 12 of the 29 proteins had not been previously reported, and their antigenicity was validated in serum from each individual patient. Results confirmed that the 12 proteins displayed nearly identical immunoglobulin G antibody levels in patients with pulmonary and extrapulmonary tuberculosis. Antigen-specific cellular interferon-γ secretion was also evaluated using a cell-based ELISPOT assay. Thirty-four of the proteins were able to induce positive interferon-γ production by peripheral blood mononuclear cells from pulmonary tuberculosis patients as judged by positive (commercial ESAT-6 antigen) and negative controls. The top 4 candidates out of the 34 proteins displayed good accuracy ranging from 50% to 80% compared with the commercial ESAT-6 antigen. Subsequent epitope examination confirmed that a pool of peptides, including a 25aa peptide from Rv1198, demonstrated significant tuberculosis-specific cellular interferon-γ production. Overall, the current study draws significant attention to novel M. tuberculosis antigens, many of which have not been previously reported. This discovery provides a large amount of useful information for the diagnosis of tuberculosis and the development of vaccines to provide protection against tuberculosis.

Project description:BackgroundNew diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB.MethodsRecombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals.ResultsA robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses.ConclusionThese results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB.

Dataset Information

Identification of specific metabolites in culture supernatant of Mycobacterium tuberculosis using metabolomics: exploration of potential biomarkers.

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Identification of specific metabolites in culture supernatant of Mycobacterium tuberculosis using metabolomics: exploration of potential biomarkers.

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