Elsevier

Tuberculosis

Volume 111, July 2018, Pages 154-160
Tuberculosis

Metabolomics of colistin methanesulfonate treated Mycobacterium tuberculosis

https://doi.org/10.1016/j.tube.2018.06.008Get rights and content

Abstract

Over the past 5 years, there has been a renewed interest in finding new compounds with anti-TB action. Colistin methanesulfonate or polymyxin E, is a possible anti-TB drug candidate, which may in future be used either alone or in combination to the current 6 month “directly observed treatment short-course” (DOTS) regimen. However its mechanism of action has to date not yet been fully explored, and only described from a histological and genomics perspective. Considering this, we used a GCxGC-TOFMS metabolomics approach and identified those metabolite markers characterising Mycobacterium tuberculosis (Mtb) cultured in the presence of colistin methanesulfonate, in order to better understand or confirm its mechanism of action. The metabolite markers identified indicated a flux in the metabolism of the colistin methanesulfonate treated Mtb towards fatty acid synthesis and cell wall repair, confirming previous reports that colistin acts by disrupting the cell wall of mycobacteria. Accompanying this, is a subsequently elevated glucose uptake, since the latter now serves as the primary energy substrate for the upregulated glyoxylate cycle, and additionally as a precursor for further fatty acid synthesis via the glycerolipid metabolic pathway. Furthermore, the elevated concentrations of those metabolites associated with pentose phosphate, valine, threonine, and pentanediol metabolism, also confirms a shift towards glucose utilization for energy production, in the colistin methanesulfonate treated Mtb.

Introduction

In 2015, an approximated 10.5 million new cases of tuberculosis (TB) were reported globally, which subsequently contributed to 1.4 million deaths [1]. Tuberculosis is caused by the infectious organism Mycobacterium tuberculosis (Mtb), a mycobacteria bacillus which mainly targets the lungs [2]. Currently, the WHO approved treatment approach entails a 6 months combination treatment approach which is called the “directly observed treatment short-course” (DOTS) regimen [3]. According to the annual WHO report, a significant improvement to current treatment strategies is going to be a challenge, however the identification of new anti-TB drug candidates and or alternative treatment regimens, might be a plausible option for speeding up treatment duration and subsequently lowering the TB prevalence globally [4,5]. Although there are currently a number of new potential anti-TB drugs undergoing phase II and III preclinical trials, delamanid and bedaquiline are the only two new anti-TB drugs to have been approved over the last 50 years. These drugs, however, are currently only used for treating adults with MDR-TB, and considered as last option medications, when no other alternatives prove to be effective [6]. Considering this, there is still urgent need for new TB drugs and alternative TB treatment approaches.

Another possible anti-TB drug candidate is the antibiotic colistin methanesulfonate (CMS), an inactive prodrug of colistin sulfate (CS), also known as polymyxin E [7]. CMS has previously been shown to have high anti-bacterial activities against P. aeruginosa, A. baumannii, and Klebsiella pneumoniae, and additionally shown to be resistant to these organisms developing drug tolerance [8]. CMS is produced via a reaction from commercially synthesised CS with formaldehyde and sodium bisulphite, resulting in the subsequent addition of a sulfomethylated group to the primary amine groups of CS [9]. The original reason for modifying CS in this manner is that the resulting CMS is considered less toxic when administered parenterally [10]. When administered, a hydrolysis reaction occurs, where CMS in an aqueous solution forms both CS and various partially sulfomethylated derivatives of CS [11]. Apart from the varying toxicity characteristics of CS and CMS, these two forms of the drug show different pharmacokinetic characteristics [[12], [13], [14]]. A study conducted by Plachouras et al. (2009), indicated that colistin concentrations increase gradually after the administration of CMS in critically ill patients, reaching a steady state after 2 days, suggesting benefits of treatment commencement with a loading dose [15]. Various colistin derivatives have also been proposed to promote first line anti-TB drug uptake, by creating pores in the outer membrane of Mtb, after binding electrostatically to the outer cell membrane lipopolysaccharides and phospholipids [16]. Very little data however exists describing the antimicrobial action of CMS against Mtb, and that which has been described to date, was attained solely from a histological or genomics approach. Metabolomics, the latest addition to the “omics” family, is defined as an unbiased identification and quantification of all metabolites present in a sample, using highly specialised analytical procedures and a statistical analysis/bioinformatics, by which the most important metabolites characterising a perturbation (or drug) can be identified [17]. In this investigation, we extracted the intracellular metabolome of Mtb cultured in the presence and absence of 32 μg/ml CMS, and analysed these extracts using a 2 dimensional gas chromatography time of flight mass spectrometry (GCxGC-TOFMS) metabolomics approach, for the purpose of identifying those metabolite markers best characterising the changes to the Mtb metabolome induced by CMS.

Section snippets

Cell culture

As described by van Breda et al. (2015), the cell cultures were prepared in the presence and absence of CMS, with slight modifications. Briefly, Mtb H37Ra ATCC 25177 (obtained from Ampath Pathology Laboratory Support Services, Centurion, Gauteng, South Africa) was swabbed onto Middlebrook 7H10 agar (Becton Dickinson, Woodmead, Gauteng, South Africa), supplemented with 0.5% v/v glycerol (Saarchem, Krugersdorp, Gauteng, South Africa), and enriched with 10% v/v oleic acid, albumin, dextrose,

Results and discussion

Fig. 1 shows clear PCA differentiation of the individually cultured Mtb samples in the presence and absence of CMS, using the collected GCxGC-TOFMS metabolomics data. The total amount of variance explained by the first two principal components (PCs) (R2X cum) was 55.9%, of which PC1 accounted for 43.4%, and PC2 accounted for 12.5%. Subsequently, by compliance with all of the following criteria: a PCA modelling power >0.5 [38], a PLS-DA VIP value > 1 [39], a t-test P-value < 0.05 and an effect

Concluding remarks

The most significant metabolite markers identified in this investigation, were the elevated concentrations of various fatty acids indicating a shift towards fatty acid synthesis and cell wall repair in the CMS treated Mtb. This is accompanied by an increase in glucose utilisation for energy and an additional flux towards the upregulation of the glyoxylate cycle (a precursor for cell wall fatty acids via the glycerolipid metabolic pathway), similarly to what was previously seen when treating Mtb

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Prof. Anton Stoltz and Prof. Ed Nardell are specifically thanked for their funding towards the cell cultures. The North West University is thanked for financial assistance of the research which forms part of a master's study.

Transparency declarations

None to declare.

References (62)

  • A. Zumla et al.

    Advances in the development of new tuberculosis drugs and treatment regimens

    Nat Rev Drug Discov

    (2013)
  • J.K. Ortwine et al.

    Colistin: understanding and applying recent pharmacokinetic advances

    Pharmacother J Hum Pharmacol Drug Ther

    (2015)
  • C.R. Catchpole et al.

    A reassessment of the in-vitro activity of colistin sulphomethate sodium

    J Antimicrob Chemother

    (1997)
  • M. Barnett et al.

    Sodium sulphomethyl derivatives of polymyxins

    Br J Pharmacol Chemother

    (1964)
  • E.G. Beveridge et al.

    Sodium sulphomethyl derivatives of polymyxins

    Br J Pharmacol Chemother

    (1967)
  • J. Li et al.

    Stability of colistin and colistin methanesulfonate in aqueous media and plasma as determined by high-performance liquid chromatography

    Antimicrob Agents Chemother

    (2003)
  • J. Li et al.

    Steady-state pharmacokinetics of intravenous colistin methanesulphonate in patients with cystic fibrosis

    J Antimicrob Chemother

    (2003)
  • J. Li et al.

    Pharmacokinetics of colistin methanesulphonate and colistin in rats following an intravenous dose of colistin methanesulphonate

    J Antimicrob Chemother

    (2004)
  • J. Li et al.

    Pharmacokinetics of colistin methanesulfonate and colistin in a critically ill patient receiving continuous venovenous hemodiafiltration

    Antimicrob Agents Chemother

    (2005)
  • D. Plachouras et al.

    Population pharmacokinetic analysis of colistin methanesulfonate and colistin after intravenous administration in critically ill patients with infections caused by gram-negative bacteria

    Antimicrob Agents Chemother

    (2009)
  • J.M. Berg et al.
  • H.I. Bax et al.

    Colistin as a potentiator of anti-TB drug activity against Mycobacterium tuberculosis

    J Antimicrob Chemother

    (2015)
  • Z. Liu et al.

    Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae

    Biotechnol Bioeng

    (2012)
  • C.-C.H. Chen et al.

    Locus of divalent cation inhibition of the bactericidal action of polymyxin B

    Antimicrob Agents Chemother

    (1972)
  • G.F. D’amato et al.

    Analisi della fluorescenza da quinacrina nel complemento cromosomico di Tragopogon porrifolius L., T. eriospermus Ten., T. crocifolius L. e di Geropogon glaber L.(Compositae)

    (1977)
  • M.E. Falagas et al.

    Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections

    Clin Infect Dis

    (2005)
  • D. Landman et al.

    Polymyxins revisited

    Clin Microbiol Rev

    (2008)
  • R.E.W. Hancock et al.

    Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies

    Nat Biotechnol

    (2006)
  • S. Ramón-García et al.

    Targeting Mycobacterium tuberculosis and other microbial pathogens using improved synthetic antibacterial peptides

    Antimicrob Agents Chemother

    (2013)
  • T.R. Sampson et al.

    Rapid killing of Acinetobacter baumannii by polymyxins is mediated by a hydroxyl radical death pathway

    Antimicrob Agents Chemother

    (2012)
  • K. Pethe et al.

    A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy

    Nat Commun

    (2010)
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