Elucidating the Antimycobacterial Mechanism of Action of Decoquinate Derivative RMB041 Using Metabolomics

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), still remains one of the leading causes of death from a single infectious agent worldwide. The high prevalence of this disease is mostly ascribed to the rapid development of drug resistance to the current anti-TB drugs, exacerbated by lack of patient adherence due to drug toxicity. The aforementioned highlights the urgent need for new anti-TB compounds with different antimycobacterial mechanisms of action to those currently being used. An N-alkyl quinolone; decoquinate derivative RMB041, has recently shown promising antimicrobial activity against Mtb, while also exhibiting low cytotoxicity and excellent pharmacokinetic characteristics. Its exact mechanism of action, however, is still unknown. Considering this, we used GCxGC-TOFMS and well described metabolomic approaches to analyze and compare the metabolic alterations of Mtb treated with decoquinate derivative RMB041 by comparison to non-treated Mtb controls. The most significantly altered pathways in Mtb treated with this drug include fatty acid metabolism, amino acid metabolism, glycerol metabolism, and the urea cycle. These changes support previous findings suggesting this drug acts primarily on the cell wall and secondarily on the DNA metabolism of Mtb. Additionally, we identified metabolic changes suggesting inhibition of protein synthesis and a state of dormancy

Qualitative serum organic acid profiles of HIV-infected individuals not on antiretroviral treatment

The first application of gas chromatography mass spectrometry (GC–MS) metabolomics to the analysis of organic acid profiles in sera of asymptomatic human immunodeficiency virus (HIV)-infected individuals (n = 18) compared to uninfected controls (n = 21), is reported here. Several organic acids are well-established diagnostic biomarkers of mitochondrial dysfunction, making the analysis of the organic acid metabolome well suited to monitoring the progressive disruption of mitochondrial structure and function during HIV infection. Using a multifaceted analytical-bioinformatics procedure, at least 10 of these metabolites could be linked to (1) disrupted mitochondrial metabolism, (2) changes in lipid metabolism and (3) oxidative stress, all of which are aberrations caused by HIV infection. Because of the role of the mitochondria in apoptosis, higher levels of this type of cell death in infected (compared to uninfected) individuals was used to support GC–MS data. This study demonstrates that mass spectrometry metabolomics detects biomarkers of mitochondrial dysfunction which could potentially be developed into indicators of HIV infection, perhaps also to monitor disease progression and the response to antiretroviral treatment.The National Research Foundationhttp://www.springerlink.com/content/1573-3882/nf201

Elucidating the Antimycobacterial Mechanism of Action of Ciprofloxacin Using Metabolomics

Abstract: In the interest of developing more effective and safer anti-tuberculosis drugs, we used a GCxGC-TOF-MS metabolomics research approach to investigate and compare the metabolic profiles of Mtb in the presence and absence of ciprofloxacin. The metabolites that best describe the differences between the compared groups were identified as markers characterizing the changes induced by ciprofloxacin. Malic acid was ranked as the most significantly altered metabolite marker induced by ciprofloxacin, indicative of an inhibition of the tricarboxylic acid (TCA) and glyoxylate cycle of Mtb. The altered fatty acid, myo-inositol, and triacylglycerol metabolism seen in this group supports previous observations of ciprofloxacin action on the Mtb cell wall. Furthermore, the altered pentose phosphate intermediates, glycerol metabolism markers, glucose accumulation, as well as the reduction in the glucogenic amino acids specifically, indicate a flux toward DNA (as well as cell wall) repair, also supporting previous findings of DNA damage caused by ciprofloxacin. This study further provides insights useful for designing network whole-system strategies for the identification of possible modes of action of various drugs and possibly adaptations by Mtb resulting in resistanc

Untargeted urine metabolomics reveals a biosignature for muscle respiratory chain deficiencies

Mitochondrial diseases are a heterogeneous group of disorders characterised by impaired mitochondrial oxidative phosphorylation system. Most often for mitochondrial disease, where no metabolic diagnostic biomarkers exist, a deficiency is diagnosed after analysing the respiratory chain enzymes (complexes I-IV) in affected tissues or by identifying one of an ever expanding number of DNA mutations. This presents a great challenge to identify cases to undergo the invasive diagnostic procedures required. An untargeted liquid chromatography mass spectrometry metabolomics approach was used to search for a metabolic biosignature that can distinguish respiratory chain deficient (RCD) patients from clinical controls (CC). A cohort of 37 ethnically diverse cases was used. Sample preparation, liquid chromatography time-of-flight mass spectrometry methods and data processing methods were standardised. Furthermore the developed methodology used reverse phase chromatography in conjunction with positive electrospray ionisation and hydrophilic interaction chromatography with negative electrospray ionisation. Urine samples of 37 patients representing two different experimental groups were analysed. The two experimental groups comprised of patients with confirmed RCDs and CC. After a variety of data mining steps and statistical analyses a list of 12 features were compiled with the ability to distinguish between patients with RCDs and CC. Although the features of the biosignature needs to be identified and the biosignature validated, this study demonstrates the value of untargeted metabolomics to identify a metabolic biosignature to possibly be applied in the selection criteria for RCDs.North-West University, Potchefstroom Campushttp://link.springer.com/journal/113062016-02-28hb201

Elucidating the antimycobacterial mechanism of action of ciprofloxacin using metabolomics

In the interest of developing more effective and safer anti-tuberculosis drugs, we used a GCxGC-TOF-MS metabolomics research approach to investigate and compare the metabolic profiles of Mtb in the presence and absence of ciprofloxacin. The metabolites that best describe the differences between the compared groups were identified as markers characterizing the changes induced by ciprofloxacin. Malic acid was ranked as the most significantly altered metabolite marker induced by ciprofloxacin, indicative of an inhibition of the tricarboxylic acid (TCA) and glyoxylate cycle of Mtb. The altered fatty acid, myo-inositol, and triacylglycerol metabolism seen in this group supports previous observations of ciprofloxacin action on the Mtb cell wall. Furthermore, the altered pentose phosphate intermediates, glycerol metabolism markers, glucose accumulation, as well as the reduction in the glucogenic amino acids specifically, indicate a flux toward DNA (as well as cell wall) repair, also supporting previous findings of DNA damage caused by ciprofloxacin. This study further provides insights useful for designing network whole-system strategies for the identification of possible modes of action of various drugs and possibly adaptations by Mtb resulting in resistance.https://www.mdpi.com/journal/microorganismsam2022Plant Production and Soil Scienc

A urinary biosignature for mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS)

We used a comprehensive metabolomics approach to study the altered urinary metabolome of two mitochondrial myopathy, encephalopathy lactic acidosis and stroke like episodes (MELAS) cohorts carrying the m.3243A > G mutation. The first cohort were used in an exploratory phase, identifying 36 metabolites that were significantly perturbed by the disease. During the second phase, the 36 selected metabolites were able to separate a validation cohort of MELAS patients completely from their respective control group, suggesting usefulness of these 36 markers as a diagnostic set. Many of the 36 perturbed metabolites could be linked to an altered redox state, fatty acid catabolism and one-carbon metabolism. However, our evidence indicates that, of all the metabolic perturbations caused by MELAS, stalled fatty acid oxidation prevailed as being particularly disturbed. The strength of our study was the utilization of five different analytical platforms to generate the robust metabolomics data reported here. We show that urine may be a useful source for disease-specific metabolomics data, linking, amongst others, altered one-carbon metabolism to MELAS. The results reported here are important in our understanding of MELAS and might lead to better treatment options for the disease.Peer reviewe

Expression of recombinant human metallothionein 2A as internal standard for mass spectrometric analysis of metallothioneins

Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2008.The induction of metallothionein (MT) expression in mitochondrial disorders has been well studied on the transcription level by means of RNA measurements in an attempt to understand and confirm the function of this protein in the deficient cells and organs (Olivier, 2004:42; Pretorius, 2006:44; Reinecke, 2004:89). However, MT expression induction still needs to be verified on protein (translation) level in order to confirm previous findings and to gain better perspective on the significance of MT expression induction. Therefore, it is necessary to use a technique that is capable of quantifying MT accurately in biological material. Due to the lack of sensitivity and selectivity of many commonly used techniques (Dabrio et al., 2002:125), it is necessary to develop a mass spectrometric based quantification technique to detect and quantify MT-2A selectively and accurately. For quantification of human MT-2A in biological material using a mass spectrometry-based method, a MT (MT-2A) standard similar to the native form but with a slightly different mass was required. Due to the lack of pure human MT standards and high cost of pure rabbit MT standards, it was decided to create a recombinant human MT-2A with different mass due to additional N-terminal amino acids. In addition, native human MT-2A is also required to develop and optimize an MS quantification technique in a future study. Therefore, pure (98 %) rabbit MT standard, which is highly similar to human MT-2A, was purchased to serve as a positive control for MS detection in this study and which can also be used to develop and optimize an MS quantification technique in a future study. An expression vector for human MT-2A was constructed with the use of recombinant DNA techniques. The correct construct was identified and characterized with PCR and verified by sequencing. This newly created expression vector was transformed into four E.coli BL21(DE3) strains to express a modified human recombinant MT-2A (MT-2AA) using induction with IPTG. This protein comprised of a full length human MT-2A sequence, but excluding the N-terminal Met and including an N-terminal His-tag. MT-2AA expression in the selected strains was extensively optimized and monitored with SDS-PAGE. Ecoli BL21 (DE3) CodonPlus-RIL cells proved to be the strain that expressed MT-2'A at the highest relative levels. Expressed MT-2'A was isolated and purified using a three step purification procedure which included heat treatment, metal chelating chromatography and RP-HPLC. Relative pure (70 %) MT-2'A was successfully obtained as confirmed with SDS-PAGE and mass spectrometry. Removal of the His-tag from MT-2'A with thrombin protease cleavage was, however, unsuccessful. In addition, it was observed that this protein was, compared to native commercially obtained MT-2A, unstable and after extensive purification still had a lower than required purity. It was concluded from this studies' results that, although it was successfully produced, this recombinant MT-2A protein would not be suitable as an internal standard for MS analysis of human MT-2A. On the other hand, rabbit MT-2E (as alternative) holds great promise as internal standard since it is stable and pure.Master

Running from depression: the antidepressant-like potential of prenatal and pre-pubertal exercise in adolescent FSL rats exposed to an early-life stressor

Objective: We aimed to answer the questions of whether early-life (perinatal and/or juvenile) exercise can induce antidepressant-like effects in a validated rodent model of depression, and whether such early-life intervention could prevent or reverse the adverse effects of early-life stress in their offspring. Methods: Male and female Flinders sensitive line rats born to a dam that exercised during gestation, or not, were either maternally separated between PND02 and 16 and weaned on PND17 or not. Half of these animals then underwent a fourteen-day low-intensity exercise regimen from PND22. Baseline depressive-like behaviour was assessed on PND21 and then reassessed on PND36, whereafter hippocampal monoamine levels, redox state markers and metabolic markers relevant to mitochondrial function were measured. Results: Pre-pubertal exercise was identified as the largest contributing factor to the observed effects, where it decreased immobility time in the FST by 6%, increased time spent in the open arms of the EPM by 9%. Hippocampal serotonin and norepinephrine levels were also increased by 35% and 26%, respectively, whilst nicotinic acid was significantly decreased. Conclusion: These findings suggest that pre-pubertal low-intensity exercise induces beneficial biological alterations that could translate into antidepressant behaviour in genetically susceptible individuals

Genetically predisposed and resilient animal models of depression reveal divergent responses to early-life adversity

Objective: Early-life adversity (ELA) is one of the strongest predictors of childhood depression that may be exacerbated by a genetic predisposition to develop depression. We therefore investigated the bio-behavioural effects of an early-life stressor in an accepted rodent model of depression. Methods: The Flinders sensitive line (FSL) and resistant line (FRL) rats were subjected to an early-life stressor, where after their bio-behavioural response during pubertal onset was evaluated. Male and female pups were maternally separated for 3 h per day from postnatal day 02 (PND02) to 17, when they were also weaned. Control animals were left undisturbed, until weaning on PND21. Depressive-like behaviour was analysed on PND21 and reassessed on PND36. Hippocampal monoamine levels, markers of oxidative stress and metabolic markers implicating mitochondrial function were also measured. Results: On PND21, the non-maternal separation and early weaning (non-MSEW) FSL rats spent 10% more time mobile than their FRL controls in the tail suspension test (TST) yet displayed increased depressive-like behaviour in the forced swim test (FST) on PND36. This depressive-like behaviour coincided with increased hippocampal norepinephrine levels, serotonin turnover and a dysfunctional redox state. Maternal separation and early weaning (MSEW) appeared to initially reduce early-life (PND21) depressive like behaviour in the TST but then induced depressive-like behaviour on PND36 and increased norepinephrine levels more profoundly in the FRL rats. Conclusion: These findings highlight the need to further investigate the stress response pathway in these animals and that the absence or presence of genetic susceptibility may influence the presentation of ELA effects