An Altered Mycobacterium tuberculosis Metabolome Induced by katG Mutations Resulting in Isoniazid Resistance

Abstract

The most common form of drug resistance found in tuberculosis (TB)-positive clinical samples is monoresistance to isoniazid. Various genomics and proteomics studies to date have investigated this phenomenon; however, the exact mechanisms relating to how this occurs, as well as the implications of this on the TB-causing organisms function and structure, are only partly understood. Considering this, we followed a metabolomics research approach to identify potential new metabolic pathways and metabolite markers, which when interpreted in context would give a holistic explanation for many of the phenotypic characteristics associated with a katG mutation and the resulting isoniazid resistance in Mycobacterium tuberculosis. In order to achieve these objectives, gas chromatography-time of flight mass spectrometry (GCxGC-TOFMS)-generated metabolite profiles from two isoniazid-resistant strains were compared to a wild-type parent strain. Principal component analyses showed clear differentiation between the groups, and the metabolites best describing the separation between these groups were identified. It is clear from the data that due to a mutation in the katG gene encoding catalase, the isoniazid-resistant strains experience increased susceptibility to oxidative stress and have consequently adapted to this by upregulating the synthesis of a number of compounds involved in (i) increased uptake and use of alkanes and fatty acids as a source of carbon and energy and (ii) the synthesis of a number of compounds directly involved in reducing oxidative stress, including an ascorbic acid degradation pathway, which to date hasn't been proposed to exist in these organisms.

Mycobacterium tuberculosis Metabolism

“…Other approaches include the analysis of sputum for both mycobacterial and host markers of disease (du Preez and Loots 2013), as well as the development of urinary metabolite signatures to track the response to chemotherapy (Mahapatra et al 2014). In addition, differential metabolite profiles have been investigated as a method to speciate pathogenic and nonpathogenic mycobacteria (Olivier and Loots 2012) and to investigate the impact of drug resistance mutations on bacterial physiology (Bisson et al 2012;Loots 2014), as discussed further below.…”

“…1). Fortunately, recent developments-particularly the application of systems biology tools such as analysis of labeled metabolic intermediates (Marrero et al 2010;Beste et al 2011Beste et al , 2013Watanabe et al 2011;Lee et al 2013) in combination with major advances in mass spectrometery -based technologies (du Preez and Loots 2013;Rhee 2013;Loots 2014)-promise unprecedented access to the biochemical state of M. tuberculosis in experimental models of infection, as well as in determining the mechanisms of action of new and existing anti-TB drugs.…”

TB or not TB? Improving the Understanding and Diagnosis of Tuberculosis through Metabolomics

“…These include mechanisms related to M. tuberculosis drug resistance [16,17] and virulence [18] including upregulation of various antioxidant pathways, the use of alkanes and fatty acids as alternative energy substrates [16,17,19], a shift in aconitase functionality toward mRNA binding and stability [17] and cell "...metabolomics has allowed for better understanding of the adaptations of Mycobacterium tuberculosis to the host's defense and vice versa... " future science group TB or not TB? Improving the understanding & diagnosis of TB through metabolomics Editorial wall remodeling [17,18]).…”

“…Apart from their diagnostic applications, various newly identified TB biomarkers have additionally contributed to the existing knowledge of the biology of the causative pathogen [16], as well as to various under lying disease mechanisms [9]. These include mechanisms related to M. tuberculosis drug resistance [16,17] and virulence [18] including upregulation of various antioxidant pathways, the use of alkanes and fatty acids as alternative energy substrates [16,17,19], a shift in aconitase functionality toward mRNA binding and stability [17] and cell "...metabolomics has allowed for better understanding of the adaptations of Mycobacterium tuberculosis to the host's defense and vice versa... " future science group TB or not TB?…”

“…Using GC-MS-identified volatile organic compound profiles, Phillips et al [13] were able to identify TB with a sensitivity of 84% and a specificity of 64.7%, and Kolk et al [14] with a sensitivity of 62%, a specificity of 84% and an accuracy of 77%. Furthermore, meta bolomics has been shown to be a valid approach for identifying markers characterizing/identifying various infectious Mycobacterium species with probabilities ranging from 72 to 100%, depending on the species of Mycobacterium, using as little as 1 × 10 3 cells [15], as well as drug-resistant strains [16,17]. Considering this, metabo lomics is proving to be a valuable tool for new TB-diagnostic biomarker detection, considering the need for such a method to not only detect TB, but to also differentiate between various infectious Mycobacterium species and drug-resistant strains, in a highly sensitive and selective manner.…”