The authors would like to thank all members of the laboratories of Dr. Barletta and Dr. Powers for helpful comments while developing the protocol. We thank Wendy Austin for helpful discussions and proofreading of the manuscript. The work described in this manuscript was funded by seed pilot grants to each investigator listed above from the University of Nebraska-Lincoln Redox Biology Center (parent grant #NCRR 2P20RR 017675, D. Becker, P.I.). In addition, we thank Dr. Ofelia Chacon for providing funds from her R21 grant (1R21AI087561-01A1) for research supplies and Mr. Halouska's partial salary support to standardize NMR techniques included in this publication.

1. Protocol Text
This protocol highlights the adaption of NMR methodology to M. tuberculosis (Class III agent). Therefore, Biosafety Level 3 (BSL3) practices need to be followed when conducting M. tuberculosis research in an annually certified laboratory. Exposure to laboratory-generated aerosols is the most important hazard encountered by personnel working with these microorganisms. The following procedures are conducted at our institution and variations may exist based on the...

<ol>
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A significant number of studies have analyzed the transcriptomic and proteomic profiles of M. tuberculosis under a variety of in vitro and in vivo conditions.11-16 Ultimately, changes in gene expression and enzyme activity lead to variations in the concentrations of small molecular weight molecules. The complete description of these compounds constitutes the metabolome. Thus, the effects of drugs and varying growth conditions on metabolic pathways may be followed by metabolomic analy...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>ADC Enrichment</td>
 <td>BD BBL Middlebrook</td>
 <td>212352</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BACS-120 Sample Changer</td>
 <td>Bruker</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bruker Avance NMR</td>
 <td>Bruker</td>
 <td>&nbsp;</td>
 <td>500 MHz</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Fisher Scientific</td>
 <td>BP1600-100</td>
 <td>Fraction V</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Allegra X-15R</td>
 <td>Benchtop</td>
 </tr>
 <tr>
 <td>Centrifuge Tubes</td>
 <td>Corning</td>
 <td>430291</td>
 <td>50 ml sterile polypropylene</td>
 </tr>
 <tr>
 <td>Cryogenic Vials</td>
 <td>Corning</td>
 <td>430488</td>
 <td>2.0 ml sterile polypropylene</td>
 </tr>
 <tr>
 <td>Cycloheximide</td>
 <td>A.G. Scientific</td>
 <td>C-1189</td>
 <td>Toxic</td>
 </tr>
 <tr>
 <td>D(+) - Glucose</td>
 <td>ACROS</td>
 <td>41095-0010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Deuterium Oxide</td>
 <td>Sigma Aldrich</td>
 <td>617385</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Erlenmeyer Flask</td>
 <td>VWR</td>
 <td>89095-266</td>
 <td>Sterile, flat base, polycarbonate, 0.22 &mu;m PTFE membrane vented cap</td>
 </tr>
 <tr>
 <td>Flash Freeze Flask</td>
 <td>VWR</td>
 <td>82018-226</td>
 <td>750 ml</td>
 </tr>
 <tr>
 <td>Freeze Dryer</td>
 <td>VWR</td>
 <td>82019-038</td>
 <td>4.5 L Benchtop</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>GibcoBRL</td>
 <td>15514-029</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Incubator</td>
 <td>New Brunswick</td>
 <td>Innova 40</td>
 <td>Benchtop shaker</td>
 </tr>
 <tr>
 <td>Lysing Matrix B</td>
 <td>MP Biomedicals</td>
 <td>6911-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lysis Machine</td>
 <td>MP Biomedicals</td>
 <td>FastPrep-24</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td>Eppendorf</td>
 <td>5415D</td>
 <td>Benchtop</td>
 </tr>
 <tr>
 <td>Microcentrifuge</td>
 <td>Beckman Coulter</td>
 <td>Microfuge 22R</td>
 <td>Benchtop</td>
 </tr>
 <tr>
 <td>Middlebrook 7H9 Broth</td>
 <td>Difco</td>
 <td>271310</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NMR tubes</td>
 <td>Norell</td>
 <td>ST500-7</td>
 <td>5mM</td>
 </tr>
 <tr>
 <td>OADC Enrichment</td>
 <td>BD BBL Middlebrook</td>
 <td>212351</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oleic Acid</td>
 <td>Sigma</td>
 <td>O1008</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium Phosphate Dibasic</td>
 <td>VWR</td>
 <td>BDH0266</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium Phosphate Monobasic</td>
 <td>VWR</td>
 <td>BDH0268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rotor - Microfuge 22R</td>
 <td>Beckman Coulter</td>
 <td>F241.5P</td>
 <td>Sealed and polypropylene</td>
 </tr>
 <tr>
 <td>Rotor - Allegra X-15R</td>
 <td>Beckman Coulter</td>
 <td>SX4750</td>
 <td>With bio-certified covers</td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Fisher Scientific</td>
 <td>S271-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium-3-trimethylsilylpropionate-2,2,3,3-D4</td>
 <td>Cambridge Isotope</td>
 <td>DLM-48</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spectrophotometer</td>
 <td>Beckman Coulter</td>
 <td>DU-530</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spectrophotometer Cuvettes</td>
 <td>LifeLINE</td>
 <td>LS-2410</td>
 <td>1.5 ml polystyrene, 2 clear sides</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Becton Dickinson</td>
 <td>309585</td>
 <td>Sterile, 3 ml Luer-Lok</td>
 </tr>
 <tr>
 <td>Syringe Filter</td>
 <td>Nalgene</td>
 <td>190-2520</td>
 <td>0.2 &mu;m sterile cellulose acetate</td>
 </tr>
 <tr>
 <td>Tween 80</td>
 <td>Fisher Scientific</td>
 <td>BP338-500</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

sample preparation of mycobacterium tuberculosis extracts for nuclear magnetic resonance metabolomic studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Watch this Scientific Journal Video about Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies at JoVE.com

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

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15.2K visualizações.  University of Nebraska-Lincoln. O perfil de metabolômica Mycobacterium tuberculosis É determinada após crescimento em culturas em caldo. As condições podem ser variadas para testar os efeitos de suplementos nutricionais, oxidantes e agentes anti-tuberculose no perfil metabólico deste microrganismo. Procedimento para a preparação de extracto é aplicável tanto para 1D 1 H e 2D 1 H- 13 Análises de C NMR.