Metabolic Profiling of Lung Granuloma in Mycobacterium tuberculosis Infected Guinea Pigs: Ex vivo H-1 Magic Angle Spinning NMR Studies

Department of Microbiology, Immunology and Pathology, Colorado State University, Campus Delivery 1682, Fort Collins, Colorado 80523-1682, United States.
Journal of Proteome Research (Impact Factor: 4.25). 07/2011; 10(9):4186-95. DOI: 10.1021/pr2003352
Source: PubMed


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.

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Available from: Bagganahalli S Somashekar, Sep 02, 2014
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    • "Major challenges for investigations of the CSF metabolome are (1) the diverse nature and low concentrations of metabolites, (2) limited availability and (3) small sample volumes (Guo et al. 2011; Stoop et al. 2010), as a result of ethical issues, such as pain and health risks, that restrict the general availability of CSF for research purposes (Geiszler et al. 2013). To date, NMR-based metabolomics of TB has been conducted on infected mice (Shin et al. 2011), guinea pigs (Somashekar et al. 2011) and humans (Zhou et al. 2013), on other forms of meningitis (Coen et al. 2005; Himmelreich et al. 2009) and a study by Subramanian et al. (2005), which selected 12 CSF metabolites for differential diagnosis of meningitis in children through in-house software diagnostics. The focus of the NMR metabolomics study of CSF reported here is directed towards the host's metabolic response to the TBM infection. "
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    ABSTRACT: Tuberculosis meningitis (TBM) is the most severe form of extra-pulmonary tuberculosis and is particularly intense in small children; there is no universally accepted algorithm for the diagnosis and substantiation of TB infection, which can lead to delayed intervention, a high risk factor for morbidity and mortality. In this study a proton magnetic resonance (1H NMR)-based metabolomics analysis and several chemometric methods were applied to data generated from lumber cerebrospinal fluid (CSF) samples from three experimental groups: (1) South African infants and children with confirmed TBM, (2) non-meningitis South African infants and children as controls, and (3) neurological controls from the Netherlands. A total of 16 NMR-derived CSF metabolites were identified, which clearly differentiated between the controls and TBM cases under investigation. The defining metabolites were the combination of perturbed glucose and highly elevated lactate, common to some other neurological disorders. The remaining 14 metabolites of the host’s response to TBM were likewise mainly energy-associated indicators. We subsequently generated a hypothesis expressed as an “astrocyte–microglia lactate shuttle” (AMLS) based on the host’s response, which emerged from the NMR-metabolomics information. Activation of microglia, as implied by the AMLS hypothesis, does not, however, present a uniform process and involves intricate interactions and feedback loops between the microglia, astrocytes and neurons that hamper attempts to construct basic and linear cascades of cause and effect; TBM involves a complex integration of the responses from the various cell types present within the CNS, with microglia and the astrocytes as main players.
    Metabolomics 10/2014; 11(4). DOI:10.1007/s11306-014-0741-z · 3.86 Impact Factor
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    • "The robustness of our models is supported by the low misclassification rates present in the LDA and by the calculated sensitivity and specificity values of our classification models, as those values observed compare favorably with the standard ante-mortem surveillance tests used in the United States [6], [9], [56], [57]. Our results were unexpected in that the intent of our work was to identify unique VOCs in the breath of M. bovis-infected cattle, based on the results of other studies exploring VOC analysis as a means of diagnosing tubercular disease in cattle [29] and other animal species [42], [58], with potential applications to humans [18], [36], [38], [39], [55]. However, our findings lead us to consider that the VOCs identified in our study represent up- or down-regulation of metabolic pathways, physiological or immune responses, or homeostatic perturbations caused by M. bovis infection. "
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    ABSTRACT: Bovine tuberculosis, caused by Mycobacterium bovis, is a zoonotic disease of international public health importance. Ante-mortem surveillance is essential for control; however, current surveillance tests are hampered by limitations affecting ease of use or quality of results. There is an emerging interest in human and veterinary medicine in diagnosing disease via identification of volatile organic compounds produced by pathogens and host-pathogen interactions. The objective of this pilot study was to explore application of existing human breath collection and analysis methodologies to cattle as a means to identify M. bovis infection through detection of unique volatile organic compounds or changes in the volatile organic compound profiles present in breath. Breath samples from 23 male Holstein calves (7 non-infected and 16 M. bovis-infected) were collected onto commercially available sorbent cartridges using a mask system at 90 days post-inoculation with M. bovis. Samples were analyzed using gas chromatography-mass spectrometry, and chromatographic data were analyzed using standard analytical chemical and metabolomic analyses, principle components analysis, and a linear discriminant algorithm. The findings provide proof of concept that breath-derived volatile organic compound analysis can be used to differentiate between healthy and M. bovis-infected cattle.
    PLoS ONE 02/2014; 9(2):e89280. DOI:10.1371/journal.pone.0089280 · 3.23 Impact Factor
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    • "Chief among these questions is the origin and molecular mechanism(s) of transport allowing mycobacterial access to aspartate inside host cells. TB lesions are enriched in aspartate, and this likely relies on global metabolic changes in immune cells during granuloma formation (Somashekar et al., 2011). How nutrients, such as aspartate, access the M. tuberculosis phagosome during infection is an intriguing issue. "
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    ABSTRACT: The tuberculosis (TB) bacillus, Mycobacterium tuberculosis, is a facultative intracellular pathogen that multiplies inside macrophages, in which it resides within a specialized compartment, the phagosome, where nutrient sources are likely limited. A number of studies provided compelling evidence that M. tuberculosis has the ability to exploit host-derived carbon sources, such as triglycerides, cholesterol and glucose, and to proliferate inside host cells (McKinney et al., 2000;Pandey and Sassetti, 2008;Daniel et al., 2011;Marrero et al., 2013). In addition to carbon, nitrogen is an essential constituent of all living organisms. As compared to carbon requirements, little is known about the nature of nitrogen-containing molecules utilized by the TB bacillus during infection. We recently discovered that nitrogen incorporation from exogenous aspartate is required for host colonization by M. tuberculosis (Gouzy et al., 2013). This study highlights, for the first time, the potential of amino acids, and aspartate in particular, as a major nitrogen reservoir supporting M. tuberculosis virulence in vivo. It also opens a series of questions to be addressed in the future.
    Frontiers in Cellular and Infection Microbiology 10/2013; 3:68. DOI:10.3389/fcimb.2013.00068 · 3.72 Impact Factor
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