9 research outputs found
Tween 80 induces a carbon flux rerouting in Mycobacterium tuberculosis
No full textAs a means to increase the growth rate and reduce aggregation, Tween 80 is routinely added to growth media during mycobacterial culturing. This detergent has, however, been associated with causing alterations to the morphology, pathogenicity and virulence of these bacteria. In an attempt to better understand the underlying mechanism of these alterations, we investigated the effect of Tween 80 on the metabolomes of a M. tuberculosis lab strain (H37Rv) and multidrug-resistant clinical strain (R179), using GC-GCxTOF-MS metabolomics. The metabolite markers identified indicated Tween 80-induced disparities in the central carbon metabolism of both strains, with an upregulation in the glyoxylate cycle, glucogenogenesis and the pentose phosphate pathway. The results also signified an increased production of mycobacterial biosynthetic precursors such as triacylglycerols, proteinogenic amino acids and nucleotide precursors, in the presence of the detergent. Collectively, these metabolome variations mimic the phenotypic changes observed when M. tuberculosis is grown in vivo, in a lipid rich environment. However, in addition to the increased availability of oleic acid as a carbon source from Tween 80, the observed variations, and the morphological changes associated with the detergent, could also be a result of an overall stress response in these bacteria. This study is the first to identify specific metabolome variations related to the addition of Tween 80 to the growth media during M. tuberculosis culturing. The consideration of these results during the method development and data interpretation phases of future metabolomics investigations will improve the quality of the analyses as well as the credibility of potential research outcomes. These results will also assist in the interpretation of research questions specifically aimed at aspects of mycobacterial metabolism, even when using other methodologies such as transcriptomics or fluxomic
Mycobacterium tuberculosis curli pili (MTP) is associated with significant host metabolic pathways in an A549 epithelial cell infection model and contributes to the pathogenicity of Mycobacterium tuberculosis
No full textIntroduction
A clear understanding of the metabolome of Mycobacterium tuberculosis and its target host cell during infection is fundamental for the development of novel diagnostic tools, effective drugs and vaccines required to combat tuberculosis. The surface-located Mycobacterium tuberculosis curli pili (MTP) adhesin forms initial contact with the host cell and is therefore important for the establishment of infection.
Objective
The aim of this investigation was to determine the role of MTP in modulating pathogen and host metabolic pathways in A549 epithelial cells infected with MTP proficient and deficient strains of M. tuberculosis.
Methods
Uninfected A549 epithelial cells, and those infected with M. tuberculosis V9124 wild-type strain, Δmtp and the mtp-complemented strains, were subjected to metabolite extraction, two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) and bioinformatic analyses. Univariate and multivariate statistical tests were used to identify metabolites that were significantly differentially produced in the WT-infected and ∆mtp-infected A549 epithelial cell models, comparatively.
Results
A total of 46 metabolites occurred in significantly lower relative concentrations in the Δmtp-infected cells, indicating a reduction in nucleic acid synthesis, amino acid metabolism, glutathione metabolism, oxidative stress, lipid metabolism and peptidoglycan, compared to those cells infected with the WT strain.
Conclusion
The absence of MTP was associated with significant changes to the host metabolome, suggesting that this adhesin is an important contributor to the pathogenicity of M. tuberculosis, and supports previous findings of its potential as a suitable drug, vaccine and diagnostic targe
Mycobacterium tuberculosis curli pili (MTP) deficiency is associated with alterations in cell wall biogenesis, fatty acid metabolism and amino acid synthesis
No full textIntroduction
In an effort to find alternative therapeutic interventions to combat tuberculosis, a better understanding of the pathophysiology of Mycobacterium tuberculosis is required. The Mycobacterium tuberculosis curli pili (MTP) adhesin, present on the surface of this pathogen, has previously been shown using functional genomics and global transcriptomics, to play an important role in establishing infection, bacterial aggregation, and modulating host response in vitro and in vivo.
Objective
This investigation aimed to determine the role of MTP in modulating the metabolism of M. tuberculosis, using mtp gene-knockout mutant and complemented strains.
Methods
Untargeted two-dimensional gas chromatography time-of-flight mass spectrometry, and bioinformatic analyses, were used to identify significant differences in the metabolite profiles among the wild-type, ∆mtp mutant and mtp-complemented strains, and validated with results generated by real-time quantitative PCR.
Results
A total of 28 metabolites were found to be significantly altered when comparing the ∆mtp mutant and the wild-type strains indicating a decreased utilisation of metabolites in cell wall biogenesis, a reduced efficiency in the breakdown of fatty acids, and decreased amino acid biosynthesis in the former strain. Comparison of the wild-type to mtp-complement, and ∆mtp to mtp-complemented strains revealed 10 and 16 metabolite differences, respectively. Real-time quantitative PCR results supported the metabolomics findings. Complementation of the ∆mtp mutant resulted in a partial restoration of MTP function.
Conclusion
The lack of the MTP adhesin resulted in various bacterial cell wall alterations and related metabolic changes. This study highlights the importance of MTP as a virulence factor and further substantiates its potential use as a suitable biomarker for the development of diagnostic tools and intervention therapeutics against T
Fatty Acid Metabolome Extraction from Mycobacterial Cells for GC-MS Metabolomics Analysis
No full textMetabolomics is becoming an increasingly popular research tool for identifying new biomarkers, which can, among other applications, be applied to elucidate various microbial growth and virulence mechanisms. Since the lipid composition of numerous microorganisms are unique and characteristic of the particular species, and in many instances also associated with several of their growth and virulence features, we developed a method for extracting the total free fatty acid metabolome from mycobacterial cells, in order to better characterize these using a gas chromatography–mass spectrometry (GC-MS) metabolomics approach. The described method can be considered an optimized Bligh–Dyer approach, since it uses the traditional solvents; chloroform, methanol and water, in a ratio of 1:2:1. However, due to the robust cell walls associated with mycobacteria, and many other microorganisms, the method was adapted to include a step which allows for the physical disruption of the cells using a vibration mill, which dramatically increases the efficiency of this approach. Hereafter, the organic phase is collected, dried, and methylated (as a derivatization step), prior to GC-MS analyse
Total Metabolome Extraction from Mycobacterial Cells for GC-MS Metabolomics Analysis
No full textOver the past 10 years, the number of metabolomics based publications in the available scientific literature has exponentially grown, a large portion of which describing new biomarkers better elucidating microbial disease mechanisms and improved diagnostics and treatment thereof. Here, we describe a metabolomics method for extracting the total metabolome (all compounds present in the microbial cell irrespective of the compound class), for analysis in a single analytical run using only one analytical instrument. This method includes disruption of robust microbial cell walls, and the precipitation of proteins and cell debris using a combination of mechanical methods and solvents. These extracts are subsequently derivatized, in order to improve the volatility of polar compounds for efficient gas chromatography-mass spectrometry (GC-MS) analysis. This methodology can be applied to all microbes, including those with robust cell walls, such as M. tuberculosis. To date, the biomarkers identified using this approach have led to improved tuberculosis (TB) diagnostics, improved TB treatment approaches, and better understanding of host–microbe interactions and associated mycobacterial genomic
Feasibility Study: Effect of Sample Pre-Treatment Procedures on Creatinine Results and Overall Implication on Downstream Diagnosis of Inherited Metabolic Disorders
No full textAbstract Introduction: Creatinine (Cr) is a chemical waste breakdown product of creatine and Cr levels can assist in diagnosing the functioning of the kidneys and it can be measured as part of basic- or comprehensive metabolic panel tests. Urinary creatinine (UCr) is often used to calculate urine analyte concentrations of metabolic panel tests, which emphasizes the need for infallibly accurate UCr results. Methods: Cr analysis was performed using an enzymatic Cr analysis kit. A total of four ERNDIM EQA urine samples with known Cr values and four samples with unknown Cr values were used in this study. Results: For the known Cr value samples, a percentage difference from the known value was calculated for each comparison. The rotated and centrifuged result comparison showed the lowest % difference from the known UCr value for known samples 1 and 4: 0.31% and 0.34% respectively. The centrifuged comparison showed lower % differences compared to those of the initial and repeat results. For the unknown UCr value samples results, standard deviations, averages and %CV (coefficient of variance) were calculated. Conclusion: This feasibility study, however small, is suggestive proof that there is indeed necessity and room for optimization when it comes to standardisation of pre-treatment procedures prior to UCr analysis
Tuberculosis is associated with sputum metabolome variations, irrespective of patient sex or HIV status: an untargeted GCxGC-TOFMS study
No full textIntroduction Various studies have identified TB-induced metabolome variations. However, in most of these studies, a large
degree of variation exists between individual patients.
Objectives To identify differential metabolites for TB, independent of patients’sex or HIV status.
Methods Untargeted GCxGC/TOF-MS analyses were applied to the sputum of 31 TB+and 197 TB- individuals. Univariate
statistics were used to identify metabolites which are significantly different between TB+and TB- individuals (a) irrespective of HIV status, and (b) with a HIV+status. Comparisons a and b were repeated for (i) all participants, (ii) males only
and (iii) females only.
Results Twenty-one compounds were significantly different between the TB+and TB- individuals within the female subgroup (11% lipids; 10% carbohydrates; 1% amino acids, 5% other and 73% unannotated), and 6 within the male subgroup
(20% lipids; 40% carbohydrates; 6% amino acids, 7% other and 27% unannotated). For the HIV+patients (TB+vs. TB-), a
total of 125 compounds were significant within the female subgroup (16% lipids; 8% carbohydrates; 12% amino acids, 6%
organic acids, 8% other and 50% unannotated), and 44 within the male subgroup (17% lipids; 2% carbohydrates; 14% amino
acids related, 8% organic acids, 9% other and 50% unannotated). Only one annotated compound, 1-oleoyl lysophosphaditic
acid, was consistently identified as a differential metabolite for TB, irrespective of sex or HIV status. The potential clinical
application of this compound should be evaluated further.
Conclusions Our findings highlight the importance of considering confounders in metabolomics studies in order to identify
unambiguous disease biomarker
The effect of Tyloxapol on the metabolome of Mycobacterium tuberculosis
No full textArticle, Faculty of Natural and Agricultural Sciences (Human Metabolomics)--Northwest University, Potchefstroom CampusThe use of detergents when culturing Mycobacterium tuberculosis (M. tuberculosis) are essential to prevent clumping. However, these detergents may influence research outcomes by impacting bacterial morphology and metabolism. This study aimed to assess the metabolome of a M. tuberculosis H37Rv strain cultured with Tyloxapol (H37RvTyloxapol), compared to a control group of H37Rv strain cultured without detergent (H37RvControl) to evaluate Tyloxapol’s suitability for metabolomic studies. Distinct metabolic alterations were observed in H37RvTyloxapol compared to H37RvControl, primarily associated with fatty acid, sugar and pentose phosphate metabolic pathways. These changes are associated with the surface stress exerted by Tyloxapol on the bacteria, prompting an adaptation of M. tuberculosis metabolism to that usually observed in stress environments. Nevertheless, the effect of Tyloxapol is less pronounced than that of a previous investigation using Tween 80, indicating its potential as the more favourable choice for culturing M. tuberculosis for metabolomic analysis, with due consideration to dosage and result interpretation
Serum metabolome changes in relation to prothrombotic state induced by combined oral contraceptives with drospirenone and ethinylestradiol
No full textThe association between hypercoagulability and use of drospirenone (DRSP) and ethinylestradiol (EE) containing
combined oral contraceptives (COCs) is an important clinical concern. We have previously reported that
the two formulations of DRSP combined with EE (namely, DRSP/20EE and DRSP/30EE) bring about a
prothrombotic state in hemostatic traits of female users. We report here the serum metabolomic changes in the
same study cohort in relation to the attendant prothrombotic state induced by COC use, thus offering new
insights on the underlying biochemical mechanisms contributing to the altered coagulatory profile with COC
use. A total of 78 healthy women participated in this study and were grouped as follows: control group not using
oral contraceptives (n = 25), DRSP/20EE group (n = 27), and DRSP/30EE group (n = 26). Untargeted metabolomics
revealed changes in amino acid concentrations, particularly a decrease in glycine and an increase in both
cysteine and lanthionine in the serum, accompanied by variations in oxidative stress markers in the COC users
compared with the controls. Of importance, this study is the first to link specific amino acid variations, serum
metabolites, and the oxidative metabolic profile with DRSP/EE use. These molecular changes could be linked to
specific biophysical coagulatory alterations observed in the same individuals. These new findings lend evidence
on the metabolomic substrates of the prothrombotic state associated with COC use in women and informs future
personalized/precision medicine research. Moreover, we underscore the importance of an interdisciplinary
approach to evaluate venous thrombotic risk associated with COC use.The National Research Foundation (NRF) (South Africa)https://home.liebertpub.com/publications/omics-a-journal-of-integrative-biology/432021-07-01am2021AnatomyPhysiolog
