Mycolic Acids Analysis Service

What are Mycolic Acids?

Mycolic acids (MAs) are the hallmark of the cell envelope of Mycobacterium tuberculosis and related species and genera. MAs are unique and complex molecular structures that are found either unbound, extractable with specific organic solvents such as esters of trehalose or glycerol, or esterifying the terminal pentaarabinofuranosyl units of arabinogalactan (AG), the polysaccharide that forms the insoluble cell wall skeleton together with peptidoglycan. Both forms probably play a crucial role in the extraordinary architecture and impermeability of the cell envelope, taking part in the two leaflets of the mycobacterial outer membrane, also referred to the mycomembrane, recently visualized using electron microscopy method. This concept has been established in Corynebacterium glutamicum, a species belonging to a genus that exhibits diverse features of the mycobacterial cell envelope, remarkably the presence of an MA-containing outer membrane.

Because Corynebacterium strains are viable in the absence of MAs, it has been proven that the outer membrane was no longer observed in C. glutamicum mutant strains devoid of MAs. Because the inhibition of MA synthesis is one of the main effects of the frontline and most efficient antitubercular drug isoniazid (INH), much attention has been paid to deciphering the chemistry and biosynthesis of MAs. The metabolic pathway of MAs represents a valuable source for recruiting latent targets for the development of novel anti-mycobacterial drugs in the alarming context of the emergence of extremely drug-resistant (XDR), multidrug-resistant (MDR), and totally drug-resistant (TDR) tuberculosis (TB). What’ more, MA-containing compounds have been associated in the past not only to numerous physiological properties of mycobacteria, such as their characteristic serpentine-like growing and ‘‘cord-forming,’’ but also to many biological properties, such as adjuvant and antineoplastic capacity of purified and crude cell wall fractions.

The last decade has seen great progress in the development of the biochemistry, structures, genetics, and regulation of MAs. Novel roles have also been discovered and attributed to MAs and their subfamilies in diverse phenomena, such as foamy macrophage formation and biofilm formation in TB granulomas.

Mycolic acids (MAs) are the hallmark of the cell envelope of Mycobacterium tuberculosis and related species and genera. MAs are unique and complex molecular structures that are found either unbound, extractable with specific organic solvents such as esters of trehalose or glycerol, or esterifying the terminal pentaarabinofuranosyl units of arabinogalactan (AG), the polysaccharide that forms the insoluble cell wall skeleton together with peptidoglycan. Both forms probably play a crucial role in the extraordinary architecture and impermeability of the cell envelope, taking part in the two leaflets of the mycobacterial outer membrane, also referred to the mycomembrane, recently visualized using electron microscopy method. This concept has been established in Corynebacterium glutamicum, a species belonging to a genus that exhibits diverse features of the mycobacterial cell envelope, remarkably the presence of an MA-containing outer membrane.

Because Corynebacterium strains are viable in the absence of MAs, it has been proven that the outer membrane was no longer observed in C. glutamicum mutant strains devoid of MAs. Because the inhibition of MA synthesis is one of the main effects of the frontline and most efficient antitubercular drug isoniazid (INH), much attention has been paid to deciphering the chemistry and biosynthesis of MAs. The metabolic pathway of MAs represents a valuable source for recruiting latent targets for the development of novel anti-mycobacterial drugs in the alarming context of the emergence of extremely drug-resistant (XDR), multidrug-resistant (MDR), and totally drug-resistant (TDR) tuberculosis (TB). What’ more, MA-containing compounds have been associated in the past not only to numerous physiological properties of mycobacteria, such as their characteristic serpentine-like growing and ‘‘cord-forming,’’ but also to many biological properties, such as adjuvant and antineoplastic capacity of purified and crude cell wall fractions.

The last decade has seen great progress in the development of the biochemistry, structures, genetics, and regulation of MAs. Novel roles have also been discovered and attributed to MAs and their subfamilies in diverse phenomena, such as foamy macrophage formation and biofilm formation in TB granulomas.

Currently, the scientists at Creative Proteomics have established a reliable and reproducible method using a highly sensitive LC-MS/MS platform for the targeted metabolomics analysis, identification, and quantification of mycolic acids in various sample types. This method can satisfy the needs of both academic and industrial studies in your lab.

Mycolic Acids Analysis in Creative Proteomics

Mycolic Acid Profiling:

• Identification and characterization of various mycolic acid types present in your samples.
• Detailed profiling to understand the structural diversity of mycolic acids.

Mycolic Acid Quantitative Analysis:

• Accurate quantification of mycolic acids using our highly sensitive LC-MS/MS platform.
• Determination of concentration levels in different sample types, including plasma, tissue, and cells.

Mycolic Acid Comparative Studies:

• Comparative analysis of mycolic acid composition across different strains or conditions.
• Identification of unique mycolic acid markers for specific mycobacterial strains.

Drug Interaction Studies:

• Examination of the effects of antitubercular drugs on mycolic acid synthesis and composition.
• Assessment of drug efficacy by monitoring changes in mycolic acid profiles.

Biochemical Pathway Elucidation:

• Detailed analysis of the biosynthetic pathways of mycolic acids.
• Identification of key enzymes and intermediates involved in mycolic acid biosynthesis.

Custom Projects:

• Tailored analysis services to meet specific research or industrial needs.
• Flexible project design to accommodate unique requirements and objectives.

Analytical Techniques for Mycolic Acids Analysis

At Creative Proteomics, we utilize the state-of-the-art liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) platform for mycolic acids analysis. This advanced analytical technique ensures high sensitivity, specificity, and accuracy, providing comprehensive insights into the complex structures and functions of mycolic acids.

Equipment Models

Thermo Scientific™ Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer:

Offers high resolution and mass accuracy, which are essential for the precise identification and quantification of mycolic acids. This instrument is particularly effective in distinguishing mycolic acids with similar masses due to its superior resolving power.

Waters Xevo TQ-XS Triple Quadrupole Mass Spectrometer:

Known for its high sensitivity and specificity, this mass spectrometer is ideal for targeted mycolic acid analysis. It provides reliable quantification even at very low concentrations, making it an excellent choice for detailed profiling.

Comprehensive Data Analysis

Software Tools:

Thermo Scientific™ TraceFinder™ Software: This software streamlines data acquisition, processing, and reporting, enhancing the efficiency and accuracy of mycolic acid analysis. It features advanced algorithms for peak identification, integration, and quantification, ensuring high data fidelity.

Waters UNIFI™ Scientific Information System: Integrates data management and analysis, providing comprehensive insights into mycolic acid profiles. This system allows for customizable analytical workflows to meet specific research needs.

Mycolic Acids Analytical Workflow

Sample Preparation:

Detailed protocols for sample extraction and preparation to ensure the integrity and accuracy of the analysis. Specific guidelines for handling different sample types (plasma, tissue, cells) to optimize the detection of mycolic acids.

LC-MS/MS Analysis:

Application of the LC-MS/MS technique to separate, identify, and quantify mycolic acids. Use of advanced mass spectrometry settings to achieve high resolution and sensitivity.

Data Processing:

Use of specialized software tools to process raw data, perform peak identification, and quantify mycolic acids. Comprehensive data analysis to interpret the results accurately and provide meaningful insights.

Reporting:

A full report including all raw data, MS/MS instrument parameters, and step-by-step calculations. Quantitative results reported in micromolar (uM) concentrations, with detailed interpretative summaries and recommendations.

Advantages of LC-MS/MS

• High Sensitivity: Detects trace levels of mycolic acids, allowing for identification of low-abundance species critical in biological studies.
• Specificity: Precisely distinguishes between structurally similar mycolic acid variants, ensuring accurate identification.
• Quantitative Accuracy: Provides robust, reproducible quantitative data with low coefficients of variation (CVs), essential for reliable measurements.
• Comprehensive Profiling: Enables detailed structural elucidation of diverse mycolic acids, including alpha-, keto-, methoxy-, and hydroxy-mycolic acids.
• Versatility: Suitable for a wide range of sample types, including plasma, tissue, and cells, making it adaptable for various research and industrial applications.
• High Resolution: Utilizes advanced mass spectrometry technology to achieve superior resolving power, essential for separating complex mixtures.
• Efficient Data Processing: Integrates with specialized software tools for streamlined data acquisition, processing, and reporting, enhancing analytical efficiency and accuracy.
• Wide Dynamic Range: Capable of analyzing mycolic acids across a broad concentration range, from trace amounts to high levels.

Sample Requirements for Mycolic Acids Analysis

Important Considerations:

• Sample Integrity and Storage Conditions: Ensure samples are collected and stored in clean, contamination-free containers. Store plasma, serum, BALF, pleural fluid, CSF, and urine at -80°C. Flash-freeze tissue samples in liquid nitrogen and store at -80°C. Keep sputum and swabs at -20°C or lower. Dry blood spots thoroughly and store in a cool, dry place at -20°C. Avoid freeze-thaw cycles to maintain sample integrity.
• Transport: Ship samples on dry ice to maintain low temperatures and prevent degradation. Seal samples tightly and protect from moisture during transport.
• Preparation: Use EDTA or heparin tubes for plasma and serum collection. Homogenize tissue samples and weigh accurately. Count cells precisely and pellet by centrifugation before freezing. Collect BALF, pleural fluid, CSF, and urine in sterile containers. Store sputum and swabs according to specified conditions. Ensure blood spots are completely dry before storage.
• Documentation: Include detailed sample information (type, volume, collection date, storage conditions). Provide relevant clinical or experimental history affecting analysis.

Report

• A full report including all raw data, MS/MS instrument parameters and step-by-step calculations will be provided (Excel and PDF formats).
• Analytes are reported as uM, with CV's generally ~10%.

In Vitro Effect of DFC-2 on Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

Journal: Journal of Microbiology and Biotechnology

Published: 2017

Background

Tuberculosis (TB) remains a significant global health concern, exacerbated by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. Conventional treatments are increasingly ineffective against these resistant strains, necessitating the development of new therapeutic agents. DFC-2 has emerged as a potential candidate due to its previously reported antitubercular activity, albeit with limited understanding of its mechanism of action, particularly its effects on mycolic acid biosynthesis.

Materials & Methods

Confirmation of Antituberculosis Activity:

Microscopic Observation Drug Susceptibility (MODS) Assay: Used to determine Minimum Inhibitory Concentrations (MIC) of DFC-2 against various M. tuberculosis strains, compared with standard drugs.

Exploring Molecular Mechanisms:

• Microarray Analysis: Employed to assess gene expression changes in M. tuberculosis H37Rv after exposure to DFC-2.
• RT-PCR Analysis: Validation of microarray results, specifically targeting genes involved in mycolic acid synthesis.

Targeted Metabolomics Service:

LC-MS/MS: Utilized for metabolomic analysis to quantify mycolic acid levels in M. tuberculosis following DFC-2 treatment.

Microscopic Observation:

Electron Microscopy: Visualized morphological changes in M. tuberculosis cells treated with DFC-2.

Results

Confirmation of Antituberculosis Activity of DFC-2

To validate the antitubercular activity previously reported for DFC-2, we conducted a Microscopic Observation Drug Susceptibility (MODS) assay. The Minimum Inhibitory Concentrations (MIC) of DFC-2 against various M. tuberculosis strains were determined and compared with standard drugs. The MICMDT of DFC-2 ranged from 0.19 to 0.39 μg/ml across all tested strains.

In vitro antitubercular activity of DFC-2 and control drugs against M. tuberculosis H37Ra, H37Rv, drug-resistant strains, and non-tubercular mycobacterial strains.

Exploring DFC-2-Induced Alterations in Gene Expression

Microarray analysis revealed significant changes in gene expression in M. tuberculosis H37Rv treated with DFC-2. At 10× MICMDT for 6 hours, 1,786 genes showed altered expression levels by 2-fold or more compared to the untreated control group. Functional classification using hypergeometric distribution highlighted "lipid biosynthesis" as the most affected category.

Validation by RT-PCR

RT-PCR analysis confirmed the downregulation of genes related to mycolic acid synthesis in M. tuberculosis upon exposure to DFC-2. Notably, genes involved in mycolic acid condensation showed significant suppression at 10× MICMDT compared to the control.

Changes in mRNA levels of M. tuberculosis genes involved in (A-E) mycolic acid synthesis and (F) heat shock protein during exposure to 1× and 10× MICTDT DFC-2 for 6 h at 37℃

Quantification of Mycolic Acid Levels by LC-MS/MS

Metabolomic analysis using LC-MS/MS demonstrated a reduction in mycolic acid production in DFC-2-treated M. tuberculosis over 5 days. This reduction paralleled the effect observed with the mycolic acid synthesis inhibitor INH, validating the impact of DFC-2 on mycolic acid biosynthesis.

Observation under Electron Microscope

Electron microscopy revealed morphological changes in M. tuberculosis cells treated with DFC-2. Compared to untreated cells, those exposed to DFC-2 displayed structural damage similar to INH-treated cells, including cell envelope disruption and loss of intracellular contents.

Comparison of Gene Expression Profiles

RT-PCR analysis of kas/FAS-II operon genes showed differential responses to DFC-2 compared to INH and ETH treatments. DFC-2 exhibited a distinct pattern of gene expression suppression related to mycolic acid synthesis, suggesting a unique mechanism of action compared to other inhibitors.

Reference

1. Kim, Sukyung, et al. "In vitro effect of DFC-2 on mycolic acid biosynthesis in Mycobacterium tuberculosis." Journal of Microbiology and Biotechnology 27.11 (2017): 1932-1941.