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Mycolic acids as diagnostic markers for tuberculosis case detection in humans and drug efficacy in mice.

Shui G, Bendt AK, Jappar IA, Lim HM, Laneelle M, Hervé M, Via LE, Chua GH, Bratschi MW, Zainul Rahim SZ, Michelle AL, Hwang SH, Lee JS, Eum SY, Kwak HK, Daffé M, Dartois V, Michel G, Barry CE, Wenk MR - EMBO Mol Med (2011)

Bottom Line: Quantification of specific precursor → fragment transitions of approximately 2000 individual mycolic acids (MAs) resulted in high analytical sensitivity and specificity.Furthermore, we quantified MA species in lung tissue of TB-infected mice and demonstrated effective clearance of MA levels following curative rifampicin treatment.Thus, our results demonstrate for the first time the feasibility and clinical relevance of direct detection of mycobacterial lipids as biomarkers of TB infection.

View Article: PubMed Central - PubMed

Affiliation: Yong Loo Lin School of Medicine, Department of Biochemistry, National University of Singapore, Singapore. guanghou_shui@nuhs.edu.sg

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MS and fingerprints of MAsMAs from bacterial cell walls were extracted and analysed by ESI/MS in negative mode.ESI/MS spectrum of MAs derived from MTB Beijing strain.Product ion analysis (MS/MS) of m/z 1164, indicating the presence of C22:0 (m/z 339), C24:0 (m/z 367) and C26:0 (m/z 395) fatty acyls in the alpha-branch. See Figs S1–4 of Supporting information for additional ESI/MS of MAs derived from Corynebacteria, Nocardia and Mycobacteria. Based on these ESI/MS and MS/MS results, we developed a method for targeted analysis using MRM. Precursor/product ion pairs (i.e. MRM transitions) selective for 670 MAs of Mycobacteria, 1024 MAs of Nocardia and 247 MAs of Corynebacteria were used to profile 33 bacterial strains. The signal intensity for each MA species was normalized to the total signal intensity of all MAs measured in each strain (percent of total MA signal), and the resulting data is expressed in the heat plot format.The inset shows an enlarged section of the α-MAs in nine mycobacterial strains (box). The triangle indicates a major α-MA C80H156O3 with C26 as the alpha-branch. The asterisk indicates the α-MA C76H148O3 with C24 as the alpha-branch. The full list of all 1942 MAs measured here, along with their MRM transitions and alpha-branch fatty acyl compositions, is shown in Table S1 of Supporting information.Single stage mass spectra of sputum from a non-TB control and from a TB patient (ESI/MS). Ion counts are indicated as counts per second (CPS).LOD of MA in sputum and in media. Serial dilutions of MTB were spiked into control sputum and medium. After MA extraction, the MA were resuspended in 120 µl of mobile phase and quantified by MRM and normalization to internal MA standard C32. Ion responses for both medium and sputum are shown using a non-linear polynomial regression curve.
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fig01: MS and fingerprints of MAsMAs from bacterial cell walls were extracted and analysed by ESI/MS in negative mode.ESI/MS spectrum of MAs derived from MTB Beijing strain.Product ion analysis (MS/MS) of m/z 1164, indicating the presence of C22:0 (m/z 339), C24:0 (m/z 367) and C26:0 (m/z 395) fatty acyls in the alpha-branch. See Figs S1–4 of Supporting information for additional ESI/MS of MAs derived from Corynebacteria, Nocardia and Mycobacteria. Based on these ESI/MS and MS/MS results, we developed a method for targeted analysis using MRM. Precursor/product ion pairs (i.e. MRM transitions) selective for 670 MAs of Mycobacteria, 1024 MAs of Nocardia and 247 MAs of Corynebacteria were used to profile 33 bacterial strains. The signal intensity for each MA species was normalized to the total signal intensity of all MAs measured in each strain (percent of total MA signal), and the resulting data is expressed in the heat plot format.The inset shows an enlarged section of the α-MAs in nine mycobacterial strains (box). The triangle indicates a major α-MA C80H156O3 with C26 as the alpha-branch. The asterisk indicates the α-MA C76H148O3 with C24 as the alpha-branch. The full list of all 1942 MAs measured here, along with their MRM transitions and alpha-branch fatty acyl compositions, is shown in Table S1 of Supporting information.Single stage mass spectra of sputum from a non-TB control and from a TB patient (ESI/MS). Ion counts are indicated as counts per second (CPS).LOD of MA in sputum and in media. Serial dilutions of MTB were spiked into control sputum and medium. After MA extraction, the MA were resuspended in 120 µl of mobile phase and quantified by MRM and normalization to internal MA standard C32. Ion responses for both medium and sputum are shown using a non-linear polynomial regression curve.

Mentions: MAs, α-alkyl-β-hydroxy branched chain fatty acids (for a molecular structure, see Fig 1B), are major envelope components of mycobacteria and targets of therapeutic intervention in TB. They are attractive diagnostic markers for mycobacterial infections for several reasons. (1) They are cell wall-associated bacterial molecules and are not synthesized in the human body. Thus, the presence of MAs in host tissue is indicative of bacterial infection. (2) The chemical diversity of MAs can be used for taxonomy. (3) MAs are involved in first-line recognition of bacteria during host–pathogen interactions, contributing to bacterial survival inside macrophages. For example CD1 receptors bind MAs and stimulate subpopulations of T cells, thus modulating the immune response to infection. (4) MAs represent a substantial mass fraction of bacterial cell walls and provide a stable and durable shell. Their hydrophobic nature renders low permeability to the cell wall and protects bacterial cells from dehydration. The MA biosynthetic machinery is thus an important target for antimycobacterial drugs (Barry et al, 1998; Ehrt & Schnappinger, 2007; Tonge, 2000). In fact, it is the chemical stability of MAs that permitted early methods for taxonomy and epidemiology of TB infections. Unfortunately, analysis of MAs is complicated by their hydrophobic properties and heterogeneous chemistry.


Mycolic acids as diagnostic markers for tuberculosis case detection in humans and drug efficacy in mice.

Shui G, Bendt AK, Jappar IA, Lim HM, Laneelle M, Hervé M, Via LE, Chua GH, Bratschi MW, Zainul Rahim SZ, Michelle AL, Hwang SH, Lee JS, Eum SY, Kwak HK, Daffé M, Dartois V, Michel G, Barry CE, Wenk MR - EMBO Mol Med (2011)

MS and fingerprints of MAsMAs from bacterial cell walls were extracted and analysed by ESI/MS in negative mode.ESI/MS spectrum of MAs derived from MTB Beijing strain.Product ion analysis (MS/MS) of m/z 1164, indicating the presence of C22:0 (m/z 339), C24:0 (m/z 367) and C26:0 (m/z 395) fatty acyls in the alpha-branch. See Figs S1–4 of Supporting information for additional ESI/MS of MAs derived from Corynebacteria, Nocardia and Mycobacteria. Based on these ESI/MS and MS/MS results, we developed a method for targeted analysis using MRM. Precursor/product ion pairs (i.e. MRM transitions) selective for 670 MAs of Mycobacteria, 1024 MAs of Nocardia and 247 MAs of Corynebacteria were used to profile 33 bacterial strains. The signal intensity for each MA species was normalized to the total signal intensity of all MAs measured in each strain (percent of total MA signal), and the resulting data is expressed in the heat plot format.The inset shows an enlarged section of the α-MAs in nine mycobacterial strains (box). The triangle indicates a major α-MA C80H156O3 with C26 as the alpha-branch. The asterisk indicates the α-MA C76H148O3 with C24 as the alpha-branch. The full list of all 1942 MAs measured here, along with their MRM transitions and alpha-branch fatty acyl compositions, is shown in Table S1 of Supporting information.Single stage mass spectra of sputum from a non-TB control and from a TB patient (ESI/MS). Ion counts are indicated as counts per second (CPS).LOD of MA in sputum and in media. Serial dilutions of MTB were spiked into control sputum and medium. After MA extraction, the MA were resuspended in 120 µl of mobile phase and quantified by MRM and normalization to internal MA standard C32. Ion responses for both medium and sputum are shown using a non-linear polynomial regression curve.
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Related In: Results  -  Collection

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fig01: MS and fingerprints of MAsMAs from bacterial cell walls were extracted and analysed by ESI/MS in negative mode.ESI/MS spectrum of MAs derived from MTB Beijing strain.Product ion analysis (MS/MS) of m/z 1164, indicating the presence of C22:0 (m/z 339), C24:0 (m/z 367) and C26:0 (m/z 395) fatty acyls in the alpha-branch. See Figs S1–4 of Supporting information for additional ESI/MS of MAs derived from Corynebacteria, Nocardia and Mycobacteria. Based on these ESI/MS and MS/MS results, we developed a method for targeted analysis using MRM. Precursor/product ion pairs (i.e. MRM transitions) selective for 670 MAs of Mycobacteria, 1024 MAs of Nocardia and 247 MAs of Corynebacteria were used to profile 33 bacterial strains. The signal intensity for each MA species was normalized to the total signal intensity of all MAs measured in each strain (percent of total MA signal), and the resulting data is expressed in the heat plot format.The inset shows an enlarged section of the α-MAs in nine mycobacterial strains (box). The triangle indicates a major α-MA C80H156O3 with C26 as the alpha-branch. The asterisk indicates the α-MA C76H148O3 with C24 as the alpha-branch. The full list of all 1942 MAs measured here, along with their MRM transitions and alpha-branch fatty acyl compositions, is shown in Table S1 of Supporting information.Single stage mass spectra of sputum from a non-TB control and from a TB patient (ESI/MS). Ion counts are indicated as counts per second (CPS).LOD of MA in sputum and in media. Serial dilutions of MTB were spiked into control sputum and medium. After MA extraction, the MA were resuspended in 120 µl of mobile phase and quantified by MRM and normalization to internal MA standard C32. Ion responses for both medium and sputum are shown using a non-linear polynomial regression curve.
Mentions: MAs, α-alkyl-β-hydroxy branched chain fatty acids (for a molecular structure, see Fig 1B), are major envelope components of mycobacteria and targets of therapeutic intervention in TB. They are attractive diagnostic markers for mycobacterial infections for several reasons. (1) They are cell wall-associated bacterial molecules and are not synthesized in the human body. Thus, the presence of MAs in host tissue is indicative of bacterial infection. (2) The chemical diversity of MAs can be used for taxonomy. (3) MAs are involved in first-line recognition of bacteria during host–pathogen interactions, contributing to bacterial survival inside macrophages. For example CD1 receptors bind MAs and stimulate subpopulations of T cells, thus modulating the immune response to infection. (4) MAs represent a substantial mass fraction of bacterial cell walls and provide a stable and durable shell. Their hydrophobic nature renders low permeability to the cell wall and protects bacterial cells from dehydration. The MA biosynthetic machinery is thus an important target for antimycobacterial drugs (Barry et al, 1998; Ehrt & Schnappinger, 2007; Tonge, 2000). In fact, it is the chemical stability of MAs that permitted early methods for taxonomy and epidemiology of TB infections. Unfortunately, analysis of MAs is complicated by their hydrophobic properties and heterogeneous chemistry.

Bottom Line: Quantification of specific precursor → fragment transitions of approximately 2000 individual mycolic acids (MAs) resulted in high analytical sensitivity and specificity.Furthermore, we quantified MA species in lung tissue of TB-infected mice and demonstrated effective clearance of MA levels following curative rifampicin treatment.Thus, our results demonstrate for the first time the feasibility and clinical relevance of direct detection of mycobacterial lipids as biomarkers of TB infection.

View Article: PubMed Central - PubMed

Affiliation: Yong Loo Lin School of Medicine, Department of Biochemistry, National University of Singapore, Singapore. guanghou_shui@nuhs.edu.sg

Show MeSH
Related in: MedlinePlus