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 Institutional Biosafety Committee's recommendations. Common personnel protective equipment will consist of a Tyvek suit, bouffant cap, booties, N95 respirator, eye protection, sleeves, and a double pair of nitrile gloves. Work involving M. tuberculosis cultures and/or manipulations with open M. tuberculosis containers is performed in the type A2 or B2 biological safety cabinet. Plastic-covered absorbent paper is placed on the working surface. All materials/supplies to be disposed or removed from the facility must be placed in two biohazard bags and decontaminated by autoclaving. Work surfaces and equipment utilized within the cabinet (FastPrep-24 lysis homogenizer, spectrophotometer, bucket with ice, etc.) must be disinfected after each work session with 1% Amphyl (tuberculocidal, bactericidal, fungicidal, and virucidal agent). M. tuberculosis cultures must be placed under double containment for transportation to larger equipment located outside of the biological safety cabinet such as freezers, incubators, centrifuges, and refrigerators. Centrifugation is conducted with enclosed safety cups and O-ring screw top tubes. For further analysis outside the BSL3 laboratory, cell-free extracts are filtered through a 0.2 &mu;m filter or microorganisms heat killed at 95 &deg;C for 15 min.2 Samples are plated to verify the absence of colony-forming units prior to removal from containment.
<ol>
 <li>Transfer 110 ml of Middlebrook 7H9 complete media containing polysorbate 80 (Tween 80 to prevent clumping) broth (MADC-TW) or a suitable medium to a 250 ml Erlenmeyer flask. Triplicate cultures are routinely grown per condition when using 13C-metabolites, and ten identical cultures are grown for 1D 1H NMR profiling. For two conditions (e.g., with and without addition of drug), double volume of culture per replicate can be grown and split into two identical cultures. All reagent and solution recipes are provided at the end of the protocol.</li>
 <li>Inoculate broth with 0.150 ml of M. tuberculosis 50% glycerol stock (allow to thaw on ice). See Note 1 below.</li>
 <li>Let the culture grow at 37 &deg;C shaking at 100 rpm for approximately 6 days (OD600 0.6-0.8). See Note 2 below.</li>
 <li>If no antibiotics, alternative additions or other treatments are needed, the cultures are ready to be harvested. If treatments are used, continue directly to Step 5. Remove a 0.5 ml sample from each flask, transfer to a microcentrifuge tube and place at 4 &deg;C for culture titration and quality control: contamination tests, phenotypic analysis, PCR tests. See Note 3 below. At this point, proceed to Step 6.</li>
 <li>Remove flasks from shaker and perform the desired treatment (e.g., add drug or metabolite). Place flasks back in the shaker and incubate for an additional period (e.g., 6-18 hr). At the end of this time, take another 1.0 ml aliquot and determine OD600. Remove a 0.5 ml sample from each flask, transfer to a microcentrifuge tube and place at 4 &deg;C for culture titration and quality control.</li>
 <li>Place cultures on ice for 5 min. After this step, leave cells on ice throughout the entire remaining protocol. Harvest cultures by centrifugation at 2,000 x g and 4 &deg;C for 15 min in 50 ml tubes using a benchtop centrifuge. Each culture will require four tubes with 25 ml each (a total of 100 ml is required to obtain adequate signal-to-noise for 2D 1H-13C NMR experiments, where only 50 ml per culture is required for 1D 1H NMR experiments).</li>
 <li>Wash each cell pellet two times with ice-cold ddH2O (approximately 15 ml the first time and 10 ml the second time) by the centrifugation parameters described above. For the second wash, combine the 10 ml aliquots into the same tube before spinning. Resuspend the cell pellet in a final volume of 1.0 ml ddH2O (need to adjust for cell pellet volume). If a single cell suspension free of clumping is desired at this step, cells can be briefly sonicated and/or passed through a 27-gauge needle three times. Alternatively, the cell pellet can be frozen at -80 &deg;C and stored until further processing. In the latter case, frozen pellets are thawed on ice prior to resuspension. Resuspend the cell pellets in a final volume of 1.0 ml ddH2O as stated above.</li>
 <li>Transfer the 1.0 ml cell suspension to a 2.0 ml screw cap tube containing Lysing Matrix B (0.1 mm silica spheres). Place the FastPrep-24 lysis homogenizer inside the biosafety cabinet. Put the samples into the sample holder, securing the retention spoke plate on top of the tube. Process the samples for 60 sec in the homogenizer at a speed setting of 6 meters/sec.</li>
 <li>Spin the samples in a microcentrifuge at 15,000 x g and 4 &deg;C for 10 min to pellet cell debris and intact cells.</li>
 <li>Remove supernatant and pass sample through a syringe filter (0.2 &mu;m) into a sterile tube. Plate 0.1 ml of sample (or a representative fraction of the sample, as for example 10%) on MADC agar to verify that there are no viable cells. Investigators may also conduct more than one filtration step and/or verify the presence of viable cells in the extracts using live-dead staining procedures so as to prevent or identify any potential biosafety concerns. Freeze samples in an ethanol-dry ice bath to store at -80 &deg;C until they are ready to be lyophilized and processed in an NMR facility.</li>
 <li>After 2 months, check plates to verify the absence of colony forming units. If no CFU's are found, samples may be taken out of the BSL3 laboratory. Further analysis is carried out in a standard NMR facility, which normally serves multi-users and operates under no specific containment requirements.</li>
 <li>Lyophilize the samples to dryness, then resuspend in 0.7 ml of NMR buffer and transfer to a microcentrifuge tube. Centrifuge for 3 min at 13,000 x g. Remove 0.6 ml and transfer to a 5 mm NMR tube. Alternatively, lyophilized samples could be mailed to an external facility as non-biohazardous regular samples.</li>
 <li>NMR data is collected immediately. Despite all precautions, active enzymes may still be present and the sample is typically not stable for long periods of time. Changes in the NMR spectrum are noticeable when the samples are left out at room temperature or at 4 &deg;C for more than 1 week. Also, NMR data collection is alternated between untreated and drug treated cultures, where samples are randomly selected from each category. This prevents unnecessary bias because a particular category had a longer delay before the NMR spectra were collected. A bias in the data would occur if the NMR spectra for all the untreated samples were collected first, and then followed by the drug treated samples. If the samples are unable to be immediately analyzed by NMR, the samples should be stored in 1 ml Eppendorf tubes at -80 &deg;C.</li>
 <li>After inserting the samples into a BACS-120 sample changer, instrument calibration, which involves a few routine steps such as lock, shim, tune, and optimization of the 90 &deg; pulse length, is required to maximize the quality of the results. A single sample is used to calibrate the 90 &deg; pulse length and tune the instrument for the remaining samples. Locking, shimming, and NMR data collection for every sample is automated using ICONNMR and gradshim.</li>
 <li>A 1D 1H NMR spectrum is collected using the Bruker zgesgp pulse sequence with water suppression using excitation sculpting. A total of 32k data points with a sweep width of 5482.5Hz, 128 scans, and 16 dummy scans are used. A 2D 1H-13C HSQC spectrum is collected using the Bruker hsqcetgp pulse sequence. A total of 2048 data points with a sweep width of 5000.0 Hz is collected along the direct 1H dimension, and 64 data points with a sweep width of 18864.9 Hz along the indirect 13C dimension. The spectrum is collected with 128 scans and 16 dummy scans to obtain good signal to noise.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/3673/3673fig1.jpg" /> 
Figure 1. A flow diagram of the experimental procedures is depicted.
NMR buffer
Stock solution of 50 mM potassium phosphate buffer with 50 &mu;M TMSP:
<ul>
 <li>2.17 g K2HPO4 (potassium phosphate dibasic)</li>
 <li>1.70 g KH2PO4 (potassium phosphate monobasic)</li>
 <li>7.86 mg Sodium-3-Trimethylsilylpropionate (TMSP-2,2,3,3-D4 (D,98%) )</li>
 <li>Dissolve in 500 ml of "100%" D2O</li>
 <li>The final solution should be at a pH 7.2 (uncorrected)</li>
</ul>
MADC-TW or MOADC-TW (1 L)
<ul>
 <li>4.7 g Middlebrook 7H9 broth base</li>
 <li>900 ml ddH2O</li>
 <li>2 ml glycerol</li>
 <li>Mix the components and autoclave for 25 min. Cool liquid to touch and add the following solutions:</li>
 <li>100 ml ADC when preparing MADC or 100 ml OADC when preparing MOADC</li>
 <li>2.5 ml 20% Tween 80 (alternatively, 1 ml of non-metabolizable 20% Tyloxapol)</li>
 <li>10 ml 1% cycloheximide</li>
</ul>
ADC (1 L)
<ul>
 <li>20 g D(+)-Glucose</li>
 <li>50 g BSA Fraction V</li>
 <li>8.5 g NaCl</li>
 <li>800 ml ddH2O</li>
 <li>Dissolve together, adjust volume to 1 L and sterilize with 0.2 &mu;m filter. Store at 4 &deg;C.</li>
</ul>
Alternatively, to prepare OADC (1L), add all components listed above plus 50 ml of 1% oleic acid (recipe below makes a 250 ml batch). Dissolve together, adjust volume to 1 L and sterilize with 0.2 &mu;m filter. Bottle needs to be wrapped in aluminum foil and store at 4 &deg;C.
<ul>
 <li>2.5 g Oleic Acid</li>
 <li>250 ml 0.2M NaOH</li>
 <li>Thaw oleic acid (solidifies upon refrigeration) by heating at 55 &deg;C for 10 min and add to NaOH solution and heat with stirring for 60 min. Store in sterile glass bottles that are wrapped in aluminum foil. Seal bottles with parafilm and store at 4 &deg;C.</li>
</ul>
If preferred, you can purchase commercial BD BBL Middlebrook ADC or OADC with catalase Enrichment.
1% Cycloheximide (100ml)
<ul>
 <li>1 g cycloheximide</li>
 <li>100 ml ddH2O</li>
</ul>
Dissolve and sterilize with 0.2 &mu;m filter. Store at 4 &deg;C. Caution: cycloheximide is toxic so handle with extreme care.
20% (v/v) Tween 80 (100ml)
<ul>
 <li>20 ml Tween 80</li>
 <li>80 ml ddH2O</li>
</ul>
Alternatively, weigh 20 g of Tween 80 (approximately 18.9 ml; density 1.06 g/ml), to prepare 20% w/v solution. Heat solution to 55 &deg;C for 30 min to dissolve, and mix completely. Sterilize liquid with a 0.2 &mu;m filter. Store the final solution at room temperature.
20% (v/v) Tyloxapol (100ml)
<ul>
 <li>20 ml Tyloxapol</li>
 <li>80 ml ddH2O</li>
</ul>
Alternatively, weigh 20 g of Tyloxapol (approximately 18.2 ml; density 1.1 g/ml), to prepare 20% w/v solution. Heat solution to 55 &deg;C for 30 min to dissolve, and mix completely. Sterilize liquid with a 0.2 &mu;m filter. Store the final solution at room temperature.
Note 1: Glycerol stocks of M. tuberculosis are prepared using the following protocol.
Stocking of M. tuberculosis
<ul>
 <li>Grow M. tuberculosis in 50 ml MADC to saturation (OD600 = 1.5 to 2.0) at 37 &deg;C under shaking conditions (100 rpm). Depending on the strain, this will take 7-14 days.</li>
 <li>Spin samples at 2,000 x g at 4 &deg;C for 15 min to pellet culture. Remove supernatant.</li>
 <li>Resuspend in 6 ml of sterile 50% glycerol.</li>
 <li>Aliquot 1.5 ml into 4 Corning cryogenic vials and label appropriately.</li>
 <li>Immediately flash freeze vials in ethanol/dry ice bath and store at -80 &deg;C.</li>
</ul>
Note 2: Inoculating strain H37Rv (-80 &deg;C stock up to 1.5 years old) into 70 ml of MADC media yields an OD600 of about 0.6 after 5 days of growth at 37 &deg;C under shaking conditions (100 rpm) in an Innova 40 Shaker.
Note 3: To test culture contamination, investigators could inoculate a culture aliquot onto standard rich medium and verify absence of growth after overnight incubation. Routinely, cultures are plated on MADC agar to observe colony morphology and examine by phase-contrast microscopy. If desired, cultures can be verified by PCR using IS6110 primers as described.3
2. Representative Results
A sample that is well prepared will yield an NMR spectrum similar to the one depicted in Figure 2. This spectrum is a representation of the M. tuberculosis wild type metabolic pool. The identified metabolites are directly or indirectly associated with the D-alanine pathway. Also, the magnitude of peak intensities is proportional to metabolite concentrations present in the cell extract. Therefore changes in peak intensities between untreated cultures, drug treatment, and mutant strains can indicate perturbations in metabolites and metabolic pathways. The M. tuberculosis 1D 1H NMR spectra were collected on a Bruker Avance 500 MHz spectrometer equipped with a triple-resonance, Z-axis gradient cryoprobe. The spectrum contains ca. 400 peaks, from which it was possible to identify and quantify 40-50 metabolites, including amino acids, nucleotide precursors, and glycolytic and citric acid intermediates. A BACS-120 sample changer with Bruker Icon software was used to automate the NMR data collection. 1D 1H NMR spectra were collected using excitation sculpting4 to efficiently remove the solvent and maintain a flat baseline, eliminating any need for baseline collection that may induce artifacts in subsequent principal component analysis (PCA) or orthogonal partial least squares discriminant analysis (OPLS-DA). A 1D 1H NMR spectrum is collected at 25 &deg;C with a spectrum width of 5482.5 Hz and 32K data points. A total of 16 dummy scans and 128 scans were used to obtain the spectrum. The ACD Labs 1D NMR processor software was used to semi-automatically process all the 1D 1H NMR spectra. The spectra were Fourier transformed, phased, and referenced to the TMSP peak (0.0 ppm). The NMRpipe software package was used to individually process the 2D 1H-13C NMR spectra and analyzed with NMRDraw. The Bruker FID data file was first converted to a file format recognizable by NMRpipe and then the spectrum was Fourier transformed, phase corrected, and zero filled. The observed NMR peaks in the 1D 1H NMR spectrum and 2D 1H-13C NMR spectrum are assigned to specific metabolites using 1H and 13C chemical shift tolerances of 0.05 and 0.50 ppm, respectively, and the Madison Metabolomics Consortium Database (MMCD),5the BioMagResBank,6 and the Human Metabolome Database.7 Specifically, the 1D and 2D NMR spectra are manually peak-picked, where the peak list of NMR chemical shifts is then uploaded to the Human Metabolome Database. The metabolites identified by the Human Metabolome Database are assigned to the NMR spectrum based on both maximizing the number of matching NMR resonances and belonging to a metabolic network. Each compound or metabolite typically has multiple C-H pairs and correspondingly multiple NMR resonances. Thus, the more of these NMR resonances that are observed in the experimental NMR spectrum, the more likely the metabolite is present. Similarly, identifying multiple metabolites associate with the same pathway increases the likelihood of correct assignments. The presence of metabolites and metabolic pathways are verified with the Kyoto Encyclopedia of Genes and Genomes (KEGG)8 and the MetaCyc databases.9 Mycobacterium smegmatis is a useful model system for M. tuberculosis and other mycobacterial pathogens. As described elsewhere, samples of M. smegmatis could also be disrupted by sonication.10
<img alt="Figure 2" src="/files/ftp_upload/3673/3673fig2.jpg" /> 
Figure 2. 1D 1H NMR spectrum of Mycobacterium tuberculosis cell extract. NMR peaks associated with representative metabolites are labeled. Abbreviations are the following: AMP, adenosine mono phosphate; &alpha;-Kg, &alpha;-ketoglutarate; Glu, Glutamate; and Ala, alanine.

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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+liquid+chromatography-mass+spectrometry+(LC-MS)+in+metabolic+profiling+and+metabolomic+studies+for+biomarker+discovery.">The future of liquid chromatography-mass spectrometry (LC-MS) in metabolic profiling and metabolomic studies for biomarker discovery.</a> Biomark Med. 1, 159-185 (2007).</li><li>Ludwig, C., Viant, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+J-resolved+NMR+spectroscopy:+review+of+a+key+methodology+in+the+metabolomics+toolbox.">Two-dimensional J-resolved NMR spectroscopy: review of a key methodology in the metabolomics toolbox.</a> Phytochem. Anal. 21, 22-32 (2010).</li><li>Simpson, R. J., Inglis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proteins and Proteomics: A Laboratory Manual. , 425-595 (2003).</li><li>Beste, D. J., McFadden, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System-level+strategies+for+studying+the+metabolism+of+Mycobacterium+tuberculosis.">System-level strategies for studying the metabolism of Mycobacterium tuberculosis.</a> Mol. Biosyst. 6, 2363-2372 (2010).</li><li>Rhee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+carbon+metabolism+in+Mycobacterium+tuberculosis:+an+unexpected+frontier.">Central carbon metabolism in Mycobacterium tuberculosis: an unexpected frontier.</a> Trends Microbiol. , (2011).</li><li>Who. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+Organization.">Health Organization.</a> Anti-tuberculosis Drug Resistance in the World: Report No. 4. , (2008).</li><li>Jassal, M., Bishai, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensively+drug-resistant+tuberculosis.">Extensively drug-resistant tuberculosis.</a> Lancet Infect Dis. 9, 19-30 (2009).</li></ol>

No conflicts of interest declared.

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><tbody><tr><td>ADC Enrichment</td><td>BD BBL Middlebrook</td><td>212352</td><td></td></tr><tr><td>BACS-120 Sample Changer</td><td>Bruker</td><td></td><td></td></tr><tr><td>Bruker Avance NMR</td><td>Bruker</td><td></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></td></tr><tr><td>Deuterium Oxide</td><td>Sigma Aldrich</td><td>617385</td><td></td></tr><tr><td>Erlenmeyer Flask</td><td>VWR</td><td>89095-266</td><td>Sterile, flat base, polycarbonate, 0.22 &amp;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></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></td></tr><tr><td>Lysis Machine</td><td>MP Biomedicals</td><td>FastPrep-24</td><td></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></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></td></tr><tr><td>Oleic Acid</td><td>Sigma</td><td>O1008</td><td></td></tr><tr><td>Potassium Phosphate Dibasic</td><td>VWR</td><td>BDH0266</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>VWR</td><td>BDH0268</td><td></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></td></tr><tr><td>Sodium-3-trimethylsilylpropionate-2,2,3,3-D4</td><td>Cambridge Isotope</td><td>DLM-48</td><td></td></tr><tr><td>Spectrophotometer</td><td>Beckman Coulter</td><td>DU-530</td><td></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 &amp;mu;m sterile cellulose acetate</td></tr><tr><td>Tween 80</td><td>Fisher Scientific</td><td>BP338-500</td><td></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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15.3K Views.  University of Nebraska-Lincoln. 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.

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

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

Pure Shift Nuclear Magnetic Resonance: a New Tool for Plant Metabolomics

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not only provides quantitative data but also permits structural identification of the metabolites present in complex mixtures.
Metabolic profiling by 1H NMR has proven useful in the study of various types of plant scenarios, which include the evaluation of crop&#160;conditions, harvest and post- harvest treatments, metabolic phenotyping, metabolic pathways, gene regulation, identification of biomarkers, chemotaxonomy, quality control, denomination of origin, among others. However, signal overlapping of the large number of resonances with expanded J-coupling multiplicities complicates the spectra analysis and its interpretation, and represents a limitation for classical 1H NMR profiling.
In the last decade, novel NMR broadband homonuclear decoupling techniques through which multiplet signals collapse into single resonance lines - commonly called Pure Shift methods - have been developed to overcome the spectra resolution problem inherent to 1H NMR classical spectra.
Here a step-by-step protocol of the plant extract preparation and the procedure to record optimal Pure Shift PSYCHE and SAPPHIRE-PSYCHE spectra in three different plant matrices - Vanilla plant&#160;leaves, potato tubers (S. tuberosum), and Cape gooseberries (P. peruviana) - is presented. The effect of the gain in resolution in metabolic identification, correlation analysis and multivariate analyses, as compared against classical spectra, is discussed.

This paper presents the use of PSYCHE and SAPPHIRE-PSYCHE in the metabolic profiling of plants and includes detailed procedures for sample preparation and optimal Pure Shift NMR spectra recording. Examples through which the gain in resolution achieved by homonuclear decoupling allows a more comprehensive understanding of the system are discussed.

Nuclear Magnetic Resonance (NMR) is one of the most powerful tools used in metabolomics. It stands as a highly accurate and reproducible method that not ...

Biochemistry

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Neuroscience

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Biology

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Gu&#233;rin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

A protocol is presented to extract the total lipid content of the cell wall of a wide range of mycobacteria. Moreover, extraction and analytical protocols of the different types of mycolic acids are shown. A thin-layer chromatographic protocol to monitor these mycobacterial compounds is also provided.

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well ...

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

The role of extracellular vesicles (EVs) in the context of bacterial infection has emerged as a new avenue for understanding microbial physiology. Specifically, Mycobacterium tuberculosis (Mtb) EVs play a role in the host-pathogen interaction and response to environmental stress. Mtb EVs are also highly antigenic and show potential as vaccine components. The most common method for purifying Mtb EVs is density gradient ultracentrifugation. This process has several limitations, including low throughput, low yield, reliance on expensive equipment, technical challenges, and it can negatively impact the resulting preparation. Size exclusion chromatography (SEC) is a gentler alternative method that combats many of the limitations of ultracentrifugation. This protocol demonstrates that SEC is effective for Mtb EV enrichment and produces high-quality Mtb EV preparations of increased yield in a rapid and scalable manner. Additionally, a comparison to density gradient ultracentrifugation by quantification and qualification procedures demonstrates the benefits of SEC. While the evaluation of EV quantity (nanoparticle tracking analysis), phenotype (transmission electron microscopy), and content (Western blotting) is tailored to Mtb EVs, the workflow provided can be applied to other mycobacteria.

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography

This protocol describes size exclusion chromatography, a facile and reproducible technique for enriching Mycobacterium tuberculosis extracellular vesicles from culture supernatants.

The role of extracellular vesicles (EVs) in the context of bacterial infection has emerged as a new avenue for understanding microbial physiology. ...

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, including metabolites, lipids, proteins, and nucleic acids, several groups have evaluated either the native or recombinant versions of bEVs for their protective potency as subunit vaccine candidates. Unlike native EVs, recombinant EVs are molecularly engineered to contain one or more immunogens of interest. Over the last decade, different groups have explored diverse approaches for generating recombinant bEVs. However, here, we report the design, construction, and enrichment of recombinant mycobacterial EVs (mEVs) in mycobacteria. Towards that, we use Mycobacterium smegmatis (Msm), an avirulent soil mycobacterium as the model system. We first describe the generation and enrichment of native EVs of Msm. Then, we describe the design and construction of recombinant mEVs that contain either mCherry, a red fluorescent reporter protein, or EsxA (Esat-6), a prominent immunogen of Mycobacterium tuberculosis. We achieve this by separately fusing mCherry and EsxA N-termini with the C-terminus of a small Msm protein Cfp-29. Cfp-29 is one of the few abundantly present proteins of MsmEVs. The protocol to generate and enrich recombinant mEVs from Msm remains identical to the generation and enrichment of native EVs of Msm.

This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis.

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

Preparing Mtb Secretome to Study Host-Pathogen Interactions

Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, apoptosis, etc., to enhance survival. While the function of a few secretory factors has been elucidated, many are yet to be characterized. Hence, investigating the Mtb secretory protein is an essential area of research. Mtb secretes proteins into the extracytoplasmic space with the help of three different secretion systems: general, twin-arginine, and specialized Type VII secretion system. One has to prepare the secretome to identify novel secretory molecules and functionally characterize the known factors. Here, we outline the method for preparing the secretome and sample preparation for mass spectrometry. Secretome preparation requires the utmost care to avoid contamination of cytosolic proteins, which can be monitored by probing with antibodies against well-characterized membrane, secretory, and cytosolic proteins. The culture filtrate thus prepared can be used for different purposes, which include investigating host immunomodulation properties.

Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis

Here, we demonstrate the preparation of Mtb culture filtrate (CF), which is the bacterial growth media that consists of proteins secreted by the bacteria. Mtb secretes proteins to modulate host signaling and immune response, which are crucial for pathogenesis. The CFs prepared are analyzed by mass spectrometry to identify all the secretory factors.

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, ...

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

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Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis