ARTICLE pubs.acs.org/jpr
Metabolic Profiling of Lung Granuloma in Mycobacterium tuberculosis Infected Guinea Pigs: Ex vivo 1H Magic Angle Spinning NMR Studies B. S. Somashekar,† Anita G. Amin,† Christopher D. Rithner,‡ JoLynn Troudt,† Randall Basaraba,† Angelo Izzo,† Dean C. Crick,† and Delphi Chatterjee*,† †
Department of Microbiology, Immunology and Pathology, Colorado State University, Campus Delivery 1682, Fort Collins, Colorado 80523-1682, United States ‡ Central Instrument Facility, Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States
bS Supporting Information ABSTRACT: A crucial and distinctive feature of tuberculosis infection is that Mycobacterium tuberculosis (Mtb) resides in granulomatous lesion at various stages of disease development and necrosis, an aspect that is little understood. We used a novel approach, applying high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) directly to infected tissues, allowing us to study the development of tuberculosis granulomas in guinea pigs in an untargeted manner. Significant up-regulation of lactate, alanine, acetate, glutamate, oxidized and the reduced form of glutathione, aspartate, creatine, phosphocholine, glycerophosphocholine, betaine, trimethylamine N-oxide, myo-inositol, scyllo-inositol, and dihydroxyacetone was clearly visualized and was identified as the infection progressed. Concomitantly, phosphatidylcholine was down-regulated. Principal component analysis of NMR data revealed clear group separation between infected and uninfected tissues. These metabolites are suggestive of utilization of alternate energy sources by the infiltrating cells that generate much of the metabolites in the increasingly necrotic and hypoxic developing granuloma through the glycolytic, pentose phosphate, and tricarboxylic acid pathways. The most relevant changes seen are, surprisingly, very similar to metabolic changes seen in cancer during tumor development. KEYWORDS: Mtb granulomas, metabolomics, HRMAS NMR, metabolic pathways, guinea pig lung
’ INTRODUCTION Mycobacterium tuberculosis (Mtb), a bacterial pathogen, causes tuberculosis (TB) that afflicts one-third of the world’s population resulting in an estimated nine million new cases of active disease and two million deaths each year.1 Upon gaining entry into human lungs, Mtb frequently persists unobtrusively causing a latent or a chronic infection that, in humans, can be divided into three phases: active phase, where Mtb actively replicates inducing host cell-mediated immunity; a chronic phase, where bacilli become dormant and patients may never develop clinical disease (latent state); and a potential reactivation phase if individuals become immunosuppressed.2 It has been shown that both at the genomic and transcriptional levels Mtb persists in the mouse lung and under in vitro conditions with a very slow growth rate.3,4 Whether these conditions actually simulate events in humans is arguable. Recent microarray analysis of infected murine lungs indicated that despite slow growth, the bacilli remain metabolically active,5 which further supports a dynamic model of bacterial persistence. The amelioration of Mtb growth in the face of an active host immune response involves replacement of sugars by fatty acids as a carbon and energy source6 and decreased energy production.4 One of the environmental factors inducing persistence is thought to be severe hypoxia presumed to occur within granulomas that r 2011 American Chemical Society
have a limited supply of oxygen7 where Mtb often resides within necrotic tissue. Under these conditions, Mtb may enter a slowlyor nonreplicating state and undergo shifts in its essential metabolic processes such as fatty acid metabolism to render current drug therapies less effective. Metabolomic strategies have distinct advantages in the identification of low molecular catabolites/anabolites in organs or biofluids in response to various pathophysiological events that can be further advanced as diagnostic biomarkers or reliable surrogates for determination of disease or health status. Assuming that Mtb strongly influences metabolism of the host and, consequently, at different stages of its life cycle may produce different metabolites influenced largely by the infiltrating cells and immunological status that comprise the developing, increasingly necrotic and hypoxic granuloma, we initiated our metabolomic studies in TB.8 The two main analytical tools that are employed for metabolomics studies are based on magnetic resonance spectroscopy and mass spectrometry.9 The unique advantage of the high resolution magic angle spinning (HRMAS) NMR technique has10 been extensively explored over the years in cancer metabolomics research. HRMAS NMR experiments Received: April 11, 2011 Published: July 07, 2011 4186
dx.doi.org/10.1021/pr2003352 | J. Proteome Res. 2011, 10, 4186–4195
Journal of Proteome Research result in highly resolved NMR spectra and at the same time maintain tissue integrity, so it can be further used for histological studies.11 In the present work, we have undertaken HRMAS NMR to study intact lung tissue metabolism at various stages of Mtb infection in the guinea pig model and correlated the metabolomic data with histological features. We identified dynamic up-regulation in specific host metabolites involved in anaerobic glycolysis, TCA cycle, anaerobic respiratory pathways, lipolysis of membrane phospholipids, osmo-regulatory, and oxidative stress metabolism. The increased metabolite concentration levels were clearly in accord with histological features such as necrosis and mineralization.
’ EXPERIMENTAL SECTION Mtb Infection in Guinea Pigs
Mtb H37Rv (TMCC#102) was grown from low-passage seed lots in Proskauer and Beck (P&B) medium containing 0.1% Tween 80 to midlog phase, and aliquots were frozen at 80 °C until used. Thirty-six female out-bred Hartley guinea pigs [∼500 g (body weight)] were purchased from Charles River Laboratories and housed in a biosafety level III animal laboratory in Colorado State University. All experimental protocols were approved by the Animal Care and Use Committee of Colorado State University and comply with NIH guidelines. Working stocks of Mtb H37Rv were diluted to 106 colony forming units (CFU/mL) in sterile distilled water, placed in the nebulizer jar, and aerosolized for 5 min. Infected animals were euthanized at 15, 30, and 60 days postinfection along with age-matched uninfected controls. The accessory lobe and half of the spleen were sampled to assess CFU numbers. Bacterial load was determined by plating organ homogenates onto nutrient Mycobacteria 7H11 agar (BD Diagnostics) supplemented with oleic acid-albumin-dextrose-catalase. Colonies were enumerated after 21 days of incubation at 37 °C. Immediately after necropsy, the lungs were harvested, snap frozen in liquid nitrogen, and stored at 80 °C under biosafety level III conditions. 1
H HRMAS NMR
All the NMR experiments on tissue sections were carried out on a Varian Inova 500 MHz NMR spectrometer equipped with a NANO probehead (Varian Inc.) In the case of infected lungs, visually identified granulomatous tissues weighing ∼20 30 mg (wet weight) were taken from various sites in the lungs. In the case of uninfected lung, tissue sections were taken randomly from animals caged for 15, 30, or 60 days. The tissue samples were rinsed with D2O and inserted in 4 mm zirconia rotors (40 μL capacity) followed by addition of ∼10 μL of D2O (Sigma Aldrich) and capped with a Kel-F plastic insert. After the sample preparation, the outer surface of the zirconia rotor was decontaminated and the rotors were kept on dry ice and transferred to the NMR facility. All of the 1H HRMAS spectra were acquired at 25 °C, the rotor spinning rate was regulated by a MAS controller (Varian Inc.) and spinning rate was also verified by the measurement of interspinning sideband distances from spectra with an accuracy of (1.0 Hz. Two sets of 1H NMR spectra were acquired with a spinning rate of 2600 Hz, a standard one-dimensional (1D) spectrum with water presaturation and a 1D Carr Purcell Meiboom Gill (CPMG) spin echo spectrum with water presaturation. The free induction decay (FID) of CPMG spin echo
ARTICLE
Table 1. Number of Guinea Pigs (N) and Excised Granulomas (x) used for HRMAS Studies to Derive Statistically Relevant Dataa uninfected
infected
15 days
N = 6, x = 25
N = 3, x = 12
30 days
N = 6, x = 19
N = 6, x = 18
60 days
N = 6, x = 15
N = 6, x = 37
a
Very few granulomas were visible in 3 out of 6 animals on day 15 of infection.
spectra were acquired with 32K data points during a 1.98 s acquisition time with a relaxation delay of 3 s; 256 transients were collected. T2 filtering was obtained with an echo time of 200 μs repeated 250 times, resulting in a 50 ms effective echo time. The acquired data were processed using TOPSPIN 2.1 software (Bruker Biospin). Resulting data were zero filled with 32K points and Fourier transformed after multiplying by an exponential window function using a line broadening function of 0.3 Hz. All of the NMR spectra were referenced to CH3 signal of lactate at δ1.33. Relative Quantification of Metabolites
In each spectrum, relative ratios of all of the quantifiable metabolites were calculated by measuring the area of individual metabolite signal with respect to the area of the anomeric proton of R-glucose at δ5.25. The intensity of glucose remained consistent in both control and infected samples throughout as noted by the visual inspection of the 1H spectra of all tissue samples. After measuring the relative intensity of all of the identified metabolites in each of the uninfected and infected spectra (Table 1), the highest ratio of a particular metabolite with respect to Glc. was considered as 100%. Relative percentage variations among these metabolites were then calculated. Statistical Analysis
Mean ( SD in each individual group was calculated using the percentage ratio values. Comparison of percentage ratios of the metabolites between groups was analyzed by Mann Whitney U-test. P values
