Functional metabolomics of the human scalp: a metabolic niche for Staphylococcus epidermidis.

Although metabolomics data acquisition and analysis technologies have become increasingly sophisticated over the past 5-10 years, deciphering a metabolite's function from a description of its structure and its abundance in a given experimental setting is still a major scientific and intellectual challenge. To point out ways to address this "data to knowledge" challenge, we developed a functional metabolomics strategy that combines state-of-the-art data analysis tools and applied it to a human scalp metabolomics data set: skin swabs from healthy volunteers with normal or oily scalp (Sebumeter score 60-120, n = 33; Sebumeter score > 120, n = 41) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), yielding four metabolomics data sets for reversed phase chromatography (C18) or hydrophilic interaction chromatography (HILIC) separation in electrospray ionization (ESI) + or - ionization mode. Following our data analysis strategy, we were able to obtain increasingly comprehensive structural and functional annotations, by applying the Global Natural Product Social Networking (M. Wang, J. J. Carver, V. V. Phelan, L. M. Sanchez, et al., Nat Biotechnol 34:828-837, 2016, https://doi.org/10.1038/nbt.3597), SIRIUS (K. Dührkop, M. Fleischauer, M. Ludwig, A. A. Aksenov, et al., Nat Methods 16:299-302, 2019, https://doi.org/10.1038/s41592-019-0344-8), and MicrobeMASST (S. ZuffaS, R. Schmid, A. Bauermeister, P. W, P. Gomes, et al., bioRxiv:rs.3.rs-3189768, 2023, https://doi.org/10.21203/rs.3.rs-3189768/v1) tools. We finally combined the metabolomics data with a corresponding metagenomic sequencing data set using MMvec (J. T. Morton, A. A. Aksenov, L. F. Nothias, J. R. Foulds, et. al., Nat Methods 16:1306-1314, 2019, https://doi.org/10.1038/s41592-019-0616-3), gaining insights into the metabolic niche of one of the most prominent microbes on the human skin, Staphylococcus epidermidis.IMPORTANCESystems biology research on host-associated microbiota focuses on two fundamental questions: which microbes are present and how do they interact with each other, their host, and the broader host environment? Metagenomics provides us with a direct answer to the first part of the question: it unveils the microbial inhabitants, e.g., on our skin, and can provide insight into their functional potential. Yet, it falls short in revealing their active role. Metabolomics shows us the chemical composition of the environment in which microbes thrive and the transformation products they produce. In particular, untargeted metabolomics has the potential to observe a diverse set of metabolites and is thus an ideal complement to metagenomics. However, this potential often remains underexplored due to the low annotation rates in MS-based metabolomics and the necessity for multiple experimental chromatographic and mass spectrometric conditions. Beyond detection, prospecting metabolites' functional role in the host/microbiome metabolome requires identifying the biological processes and entities involved in their production and biotransformations. In the present study of the human scalp, we developed a strategy to achieve comprehensive structural and functional annotation of the metabolites in the human scalp environment, thus diving one step deeper into the interpretation of "omics" data. Leveraging a collection of openly accessible software tools and integrating microbiome data as a source of functional metabolite annotations, we finally identified the specific metabolic niche of Staphylococcus epidermidis, one of the key players of the human skin microbiome.

MIAMI--a tool for non-targeted detection of metabolic flux changes for mode of action identification.

SUMMARY: Mass isotopolome analysis for mode of action identification (MIAMI) combines the strengths of targeted and non-targeted approaches to detect metabolic flux changes in gas chromatography/mass spectrometry datasets. Based on stable isotope labeling experiments, MIAMI determines a mass isotopomer distribution-based (MID) similarity network and incorporates the data into metabolic reference networks. By identifying MID variations of all labeled compounds between different conditions, targets of metabolic changes can be detected. AVAILABILITY AND IMPLEMENTATION: We implemented the data processing in C++17 with Qt5 back-end using MetaboliteDetector and NTFD libraries. The data visualization is implemented as web application. Executable binaries and visualization are freely available for Linux operating systems, the source code is licensed under General Public License version 3.

Framework for the quality assurance of 'omics technologies considering GLP requirements.

'Omics technologies are gaining importance to support regulatory toxicity studies. Prerequisites for performing 'omics studies considering GLP principles were discussed at the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) Workshop Applying 'omics technologies in Chemical Risk Assessment. A GLP environment comprises a standard operating procedure system, proper pre-planning and documentation, and inspections of independent quality assurance staff. To prevent uncontrolled data changes, the raw data obtained in the respective 'omics data recording systems have to be specifically defined. Further requirements include transparent and reproducible data processing steps, and safe data storage and archiving procedures. The software for data recording and processing should be validated, and data changes should be traceable or disabled. GLP-compliant quality assurance of 'omics technologies appears feasible for many GLP requirements. However, challenges include (i) defining, storing, and archiving the raw data; (ii) transparent descriptions of data processing steps; (iii) software validation; and (iv) ensuring complete reproducibility of final results with respect to raw data. Nevertheless, 'omics studies can be supported by quality measures (e.g., GLP principles) to ensure quality control, reproducibility and traceability of experiments. This enables regulators to use 'omics data in a fit-for-purpose context, which enhances their applicability for risk assessment.

In silico metabolic network analysis of Arabidopsis leaves.

BACKGROUND: During the last decades, we face an increasing interest in superior plants to supply growing demands for human and animal nutrition and for the developing bio-based economy. Presently, our limited understanding of their metabolism and its regulation hampers the targeted development of desired plant phenotypes. In this regard, systems biology, in particular the integration of metabolic and regulatory networks, is promising to broaden our knowledge and to further explore the biotechnological potential of plants. RESULTS: The thale cress Arabidopsis thaliana provides an ideal model to understand plant primary metabolism. To obtain insight into its functional properties, we constructed a large-scale metabolic network of the leaf of A. thaliana. It represented 511 reactions with spatial separation into compartments. Systematic analysis of this network, utilizing elementary flux modes, investigates metabolic capabilities of the plant and predicts relevant properties on the systems level: optimum pathway use for maximum growth and flux re-arrangement in response to environmental perturbation. Our computational model indicates that the A. thaliana leaf operates near its theoretical optimum flux state in the light, however, only in a narrow range of photon usage. The simulations further demonstrate that the natural day-night shift requires substantial re-arrangement of pathway flux between compartments: 89 reactions, involving redox and energy metabolism, substantially change the extent of flux, whereas 19 reactions even invert flux direction. The optimum set of anabolic pathways differs between day and night and is partly shifted between compartments. The integration with experimental transcriptome data pinpoints selected transcriptional changes that mediate the diurnal adaptation of the plant and superimpose the flux response. CONCLUSIONS: The successful application of predictive modelling in Arabidopsis thaliana can bring systems-biological interpretation of plant systems forward. Using the gained knowledge, metabolic engineering strategies to engage plants as biotechnological factories can be developed.

Novel Approach for High-Throughput Metabolic Screening of Whole Plants by Stable Isotopes.

Here, we demonstrate whole-plant metabolic profiling by stable isotope labeling and combustion isotope-ratio mass spectrometry for precise quantification of assimilation, translocation, and molecular reallocation of (13)CO2 and (15)NH4NO3 The technology was applied to rice (Oryza sativa) plants at different growth stages. For adult plants, (13)CO2 labeling revealed enhanced carbon assimilation of the flag leaf from flowering to late grain-filling stage, linked to efficient translocation into the panicle. Simultaneous (13)CO2 and (15)NH4NO3 labeling with hydroponically grown seedlings was used to quantify the relative distribution of carbon and nitrogen. Two hours after labeling, assimilated carbon was mainly retained in the shoot (69%), whereas 7% entered the root and 24% was respired. Nitrogen, taken up via the root, was largely translocated into the shoot (85%). Salt-stressed seedlings showed decreased uptake and translocation of nitrogen (69%), whereas carbon metabolism was unaffected. Coupled to a gas chromatograph, labeling analysis provided enrichment of proteinogenic amino acids. This revealed significant protein synthesis in the panicle of adult plants, whereas protein biosynthesis in adult leaves was 8-fold lower than that in seedling shoots. Generally, amino acid enrichment was similar among biosynthetic families and allowed us to infer labeling dynamics of their precursors. On this basis, early and strong (13)C enrichment of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway intermediates indicated high activity of these routes. Applied to mode-of-action analysis of herbicides, the approach showed severe disturbance in the synthesis of branched-chain amino acids upon treatment with imazapyr. The established technology displays a breakthrough for quantitative high-throughput plant metabolic phenotyping.

Structure elucidation of cyclic pyoverdins and examination of rearrangement reactions in MS/MS experiments by determination of exact product ion masses.

Structure elucidation of naturally occurring linear and cyclic peptidic compounds can be complicated by rearrangement reactions induced upon collision activation (CA) when parts of the molecule migrate, suggesting incorrect substitution patterns. Such complex rearrangements are examined and discussed for two iron complexing compounds produced by the bacterial genus Pseudomonas (so-called pyoverdins). Various MS2- and MS3-product ion experiments were performed using a quadrupole-ion trap (QIT) at low resolution and a FT-ICR at high resolution allowing accurate mass determinations. The results of the multidimensional study confirm the proposed processes. On the basis of the series of tandem-MS experiments the structure of a new pyoverdin from a P. fluorescens strain [PVD(D47)] is deduced.

Structural characterization of sulfadiazine metabolites using H/D exchange combined with various MS/MS experiments.

Two major metabolites and one minor metabolite of sulfadiazine were found in pig manure, using a special combination of different MS techniques like parent and product ion scans, H/D exchange, accurate mass measurement, and MS/MS experiments with substructures. N4-acetylsulfadiazine and 4-hydroxysulfadiazine were identified as major metabolites. N4-acetylsulfadiazine could be verified by H/D exchange and comparison with product ion spectra of a synthetic reference compound. In the case of 4-hydroxysulfadiazine, the majority of possible isomers could be discounted after H/D exchange. Substructure-specific MS/MS experiments with fragment ions and comparison with product ion spectra of two references revealed the presence of 4-hydroxysulfadiazine. The minor metabolite was characterized to some degree using H/D exchange and tandem mass spectrometry in combination with a high-resolution time of flight mass spectrometer. The aminopyrimidine moiety contained an additional modification with a likely elemental composition of C2H4O and no further acidic hydrogen.

Liquid chromatography with triple-quadrupole and quadrupole-time-of-flight mass spectrometry for the determination of micro-constituents - a comparison.

The potential of liquid chromatography with triple-quadrupole mass spectrometry (LC-QqQ MS) was compared to that of quadrupole time-of-flight mass spectrometry (LC-Q-ToF MS) for the determination of microconstituents. Three applications were studied: (1) the ng/l quantification of five human drugs in surface water and waste-water effluents; (2) the quantification and confirmation of three corticosteroids in bovine urine at concentrations of 1-100 microg/l, and; (3) the confirmation of nicotine in rat plasma. In all cases, the criteria of the EU Commission Decision 2002/657/EC were followed (for confirmation analysis two MS/MS ions were monitored, and the ratio of their abundances were calculated and compared with those of standards). With both techniques fully satisfactory results were obtained in almost all instances. That is, unequivocal confirmation according to the most stringent EU criteria, those for "illegal compounds", was possible.One main advantage of LC-Q-ToF MS is that for identification and confirmation purposes, full MS/MS spectra are available after a single injection: no second injection, as required with QqQ MS, is needed. As well as the increased efficiency, the enhanced selectivity due to the impressive mass selectivity of LC-Q-ToF MS must be emphasized, which allows accurate masses of fragment ions to be calculated. Method characteristics such as linear dynamic range and repeatability were found to be essentially the same for both techniques, but LC-QqQ MS has the advantage that its detection limits are somewhat lower.

Liquid chromatography with triple-quadrupole or quadrupole-time of flight mass spectrometry for screening and confirmation of residues of pharmaceuticals in water.

LC-MS-MS has been performed with triple-quadrupole (QqQ) and quadrupole-time of flight (Q-ToF) instruments and has been used for screening and confirmation of pharmaceuticals in surface, drinking, and ground water. Screening was based on monitoring of one specific MS-MS ion of the target compounds. Confirmation of the identity of the pharmaceuticals was based either on the monitoring of two specific MS-MS ions and calculation of the ratio of their intensities, or on the exact masses of MS-MS product ions obtained for a molecular ion by use of LC-Q-ToF MS. The set of pharmaceuticals included four analgesics (acetylsalicylic acid, diclofenac, ibuprofen, and paracetamol), three antibiotics (sulfamethoxazole, erythromycin, and chloramphenicol), five blood-lipid regulators and beta-blockers (fenofibrate, bezafibrate, clofibric acid, bisoprolol, and metoprolol), and the anti-epileptic drug carbamazepine. Limits of quantification ranged from 5 to 25 ng L(-1). Fifty-six samples were analysed and residues of the pharmaceuticals were detected in almost all surface and groundwater and in some drinking water samples. The identity of the compounds could be confirmed by use of both QqQ- and Q-ToF-based LC-MS-MS. However, the latter technique has the distinct advantage that a large number of pharmaceuticals can be screened and confirmed at low concentrations (1-100 ng L(-1)) in one run.

The pyoverdin of Pseudomonas fluorescens G173, a novel structural type accompanied by unexpected natural derivatives of the corresponding ferribactin.

The siderophores produced by Pseudomonas fluorescens G173 are unusual in several respects. So far all pyoverdins with a C-terminal cyclopeptidic substructure have in common that the epsilon-amino group of an in-chain Lys is bound amidically to the carboxyl group of a C-terminal Ser or Thr and that N5-formyl-N5-hydroxy Orn (FoOHOrn) is the next amino acid after Lys. FoOHOrn may (cyclotetrapeptidic structures) be or may not (cyclotripeptidic structures) be followed by a further amino acid. In the pyoverdin described here Orn instead of Lys is the amino acid forming the cycle, FoOHOrn is replaced by AcOHOrn which does not follow the branching Orn but is the penultimate amino acid and finally the last amino acid is Asp. The producing strain which had been classified as Pseudomonas fluorescens may well be a new species. Pyoverdins are frequently accompanied by ferribactins which are considered to be their biogenetic precursors. They always have the same amino acid chain as the co-occurring pyoverdins but the pyoverdin chromophore is replaced by a condensation product of L-Dab and D-Tyr with the amino group of Tyr bound to the gamma-carboxyl group of Glu. A ferribactin having these structural characteristics is produced by the investigated strain, but it is accompanied by derivatives where the alpha-amino group of Glu is partially or completely transformed into a hydroxamic acid by substitution with a hydroxyl and/or acetyl group.

Vanadium interferes with siderophore-mediated iron uptake in Pseudomonas aeruginosa.

Vanadium is a metal that under physiological conditions can exist in two oxidation states, V(IV) (vanadyl ion) and V(V) (vanadate ion). Here, it was demonstrated that both ions can form complexes with siderophores. Pseudomonas aeruginosa produces two siderophores under iron-limiting conditions, pyoverdine (PVD) and pyochelin (PCH). Vanadyl sulfate, at a concentration of 1-2 mM, strongly inhibited growth of P. aeruginosa PAO1, especially under conditions of severe iron limitation imposed by the presence of non-utilizable Fe(III) chelators. PVD-deficient mutants were more sensitive to vanadium than the wild-type, but addition of PVD did not stimulate their growth. Conversely, PCH-negative mutants were more resistant to vanadium than the wild-type strain. Both siderophores could bind and form complexes with vanadium after incubation with vanadyl sulfate (1:1, in the case of PVD; 2:1, in the case of PCH). Although only one complex with PVD, V(IV)-PVD, was found, both V(IV)- and V(V)-PCH were detected. V-PCH, but not V-PVD, caused strong growth reduction, resulting in a prolonged lag phase. Exposure of PAO1 cells to vanadium induced resistance to the superoxide-generating compound paraquat, and conversely, exposure to paraquat increased resistance to V(IV). Superoxide dismutase (SOD) activity of cells grown in the presence of V(IV) was augmented by a factor of two. Mutants deficient in the production of Fe-SOD (SodB) were particularly sensitive to vanadium, whilst sodA mutants deficient for Mn-SOD were only marginally affected. In conclusion, it is suggested that V-PCH catalyses a Fenton-type reaction whereby the toxic superoxide anion O(2)- is generated, and that vanadium compromises PVD utilization.