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

Summary

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.

Abstract

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 °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.

Protocol

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 r....

Discussion

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.......

Materials

List of materials used in this article

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