One-dimensional 1H NMR Spectra Research Articles

Estimation of conformational entropy for ionic liquids from NMR spectroscopy

Abstract Conformational entropy (Sconf) plays a key role in the low melting points of ionic liquids. In this study, a nuclear magnetic resonance (NMR)-based approach was developed to experimentally estimate Sconf of 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide. J-coupling constants from the 1D 1H NMR and 2D spectra of the butyl group in the cation were analyzed using the Karplus equation optimized by density functional theory calculations. The obtained conformational population and entropy values coincided reasonably well with previously reported results, indicating the usefulness of the NMR approach.

Geographical discrimination of Asian red pepper powders using 1H NMR spectroscopy and deep learning-based convolution neural networks

This study investigated an innovative approach to discriminate the geographical origins of Asian red pepper powders by analyzing one-dimensional 1H NMR spectra through a deep learning-based convolution neural network (CNN). 1H NMR spectra were collected from 300 samples originating from China, Korea, and Vietnam and used as input data. Principal component analysis − linear discriminant analysis and support vector machine models were employed for comparison. Bayesian optimization was used for hyperparameter optimization, and cross-validation was performed to prevent overfitting. As a result, all three models discriminated the origins of the test samples with over 95 % accuracy. Specifically, the CNN models achieved a 100 % accuracy rate. Gradient-weighted class activation mapping analysis verified that the CNN models recognized the origins of the samples based on variations in metabolite distributions. This research demonstrated the potential of deep learning-based classification of 1H NMR spectra as an accurate and reliable approach for determining the geographical origins of various foods.

Grazing affects metabolic pattern of individual cow milk.

Effective traceability tools able to characterize milk from pasture are important to safeguard low-input farming systems, niche dairy products, and local traditions. The aims of the present study were to investigate the ability of proton nuclear magnetic resonance (1H NMR) spectroscopy to discriminate between milk produced from cows before and after the beginning of the grazing season, and to assess the effects of grazing on milk metabolites. The research trial involved a single alpine holding with 72 lactating cows. Individual milks were repeatedly sampled from the same animals before (i.e., d -3 and -1) and after (i.e., d 2, 3, 7, 10, and 14) the onset of the grazing period. One-dimensional 1H NMR spectra of milk extracts were collected through a Bruker spectrometer. Random forest discriminant analysis was applied to 1H NMR spectra to predict the period of collection for each sample. Data concerning the relative abundance of milk metabolites were analyzed through a linear mixed model, which included the fixed effects of period of sampling, cow breed, stage of lactation, and parity, and the random effect of cow nested within breed. The random forest model exhibited great accuracy (93.1%) in discriminating between samples collected on d -3, -1, 2, and 3 and those collected on d 7, 10, and 14. Univariate analysis performed on the 40 detected metabolites highlighted that milk samples from pasture had lower levels of 14 compounds (with fumarate being the most depressed metabolite) and greater levels of 15 compounds (with methanol and hippurate being the most elevated metabolites). Results indicate that milk 1H NMR spectra are promising to identify milk produced in different conditions. Also, our study highlights that grazing is associated with significant changes of milk metabolic profile, suggesting the potential use of several metabolites as indicators of farm management.

Introducing Molecular Structural Analysis Using a Guided Systematic Approach Combined with an Interactive Multiplatform Web Application

Performing the identification of organic chemical compounds from a set of spectroscopic data gives the opportunity to students to develop critical thinking and problem-solving skills. In this context, we developed a student-centered methodology for teaching molecular structural analysis to first-year undergraduate students. This systematic approach was implemented during classroom-training sessions and complemented by a home-based training program. Home-based activities involved a multiplatform Web-based application (ULg Spectra) combined with guided inquiries. ULg Spectra offers fully interactive tutorial/drill materials relying on mass, infrared, and nuclear magnetic resonance spectra (i.e., one-dimensional 1H NMR and 13C NMR spectra and two-dimensional 1H–13C HSQC spectra). A survey indicates that the vast majority of students valued the ULg Spectra application combined with guided inquiries, especially in terms of usability and usefulness. This approach prompted them to actively engage in problem solving, and student autonomy was improved. Statistical data demonstrated that low-, medium-, and high-training students’ groups showed increasing performance in the final exam. Interestingly, a statistically significant increase in final grades and success rate was also observed compared to previous years.

Improved lipid mixtures profiling by 1H NMR using reference lineshape adjustment and deconvolution techniques

Analysisof one-dimensional 1H NMR spectra of complex mixtures, such as lipids from natural extracts, is hampered by the small spectral width leading to a great number of overlapped signals. Additional complications including lineshape broadening and distortion may occur due to magnetic field inhomogeneity. Quantitation of such spectra is therefore challenging. We present in this work a quantitation approach based on deconvolution after correction of spectra by means of reference lineshape adjustment (RLA), also known as reference deconvolution. Spectral fit and precision obtained on deconvoluted peaks were used as indicators to iteratively improve the deconvolution process. This approach was tested on 1H NMR spectra of olive oil samples and allowed extraction of 77 peaks (available as peak intensities or areas), whereas spectral integration afforded 5 variables when only well-resolved signals were considered and 29 variables when a bucket around each discernible peak was integrated. Deconvoluted peak intensities and areas were obtained with improved precision after RLA of raw spectra. The use of these spectral variables as predictors in multivariate statistical analysis enhanced the classification of olive oil samples according to the altitude of the olive field or to the color of the olive drupes. The same variables allowed quantitation of oleic, palmitoleic, and vaccenic acids within triacylglycerols, which was not possible by 1H NMR, and improved quantitation of linoleic and linolenic acids. These results proved the high potential of the presented approach in the characterization and authentication of complex mixtures by 1H NMR spectroscopy.

A putative G-quadruplex structure in the proximal promoter of VEGFR-2 has implications for drug design to inhibit tumor angiogenesis

Tumor angiogenesis is mainly regulated by vascular endothelial growth factor (VEGF) produced by cancer cells. It is active on the endothelium via VEGF receptor 2 (VEGFR-2). G-quadruplexes are DNA secondary structures formed by guanine-rich sequences, for example, within gene promoters where they may contribute to transcriptional activity. The proximal promoter of VEGFR-2 contains a G-quadruplex, which has been suggested to interact with small molecules that inhibit VEGFR-2 expression and thereby tumor angiogenesis. However, its structure is not known. Here, we determined its NMR solution structure, which is composed of three stacked G-tetrads containing three syn guanines. The first guanine (G1) is positioned within the central G-tetrad. We also observed that a noncanonical, V-shaped loop spans three G-tetrad planes, including no bridging nucleotides. A long and diagonal loop, which includes six nucleotides, connects reversal double chains. With a melting temperature of 54.51 °C, the scaffold of this quadruplex is stabilized by one G-tetrad plane stacking with one nonstandard bp, G3-C8, whose bases interact with each other through only one hydrogen bond. In summary, the NMR solution structure of the G-quadruplex in the proximal promoter region of the VEGFR-2 gene reported here has uncovered its key features as a potential anticancer drug target.

NMR metabolomics of fibroblasts with inherited mitochondrial Complex I mutation reveals treatment-reversible lipid and amino acid metabolism alterations

IntroductionElucidating molecular alterations due to mitochondrial Complex I (CI) mutations may help to understand CI deficiency (CID), not only in mitochondriopathies but also as it is caused by drugs or associated to many diseases.ObjectivesCID metabolic expression was investigated in Leber’s hereditary optic neuropathy (LHON) caused by an inherited mutation of CI.MethodsNMR-based metabolomics analysis was performed in intact skin fibroblasts from LHON patients. It used several datasets: one-dimensional 1H-NMR spectra, two-dimensional 1H-NMR spectra and quantified metabolites. Spectra were analysed using orthogonal partial least squares-discriminant analysis (OPLS-DA), and quantified metabolites using univariate statistics. The response to idebenone (IDE) and resveratrol (RSV), two agents improving CI activity and mitochondrial functions was evaluated.ResultsLHON fibroblasts had decreased CI activity (− 43%, p < 0.01). Metabolomics revealed prominent alterations in LHON including the increase of fatty acids (FA), polyunsaturated FA and phosphatidylcholine with a variable importance in the prediction (VIP) > 1 in OPLS-DA, p < 0.01 in univariate statistics, and the decrease of amino acids (AA), predominantly glycine, glutamate, glutamine (VIP > 1) and alanine (VIP > 1, p < 0.05). In LHON, treatment with IDE and RSV increased CI activity (+ 40 and + 44%, p < 0.05). IDE decreased FA, polyunsaturated FA and phosphatidylcholine (p < 0.05), but did not modified AA levels. RSV decreased polyunsaturated FA, and increased several AA (VIP > 1 and/or p < 0.05).ConclusionLHON fibroblasts display lipid and amino acid metabolism alterations that are reversed by mitochondria-targeted treatments, and can be related to adaptive changes. Findings bring insights into molecular changes induced by CI mutation and, beyond, CID of other origins.

ULg Spectra: An Interactive Software Tool To Improve Undergraduate Students’ Structural Analysis Skills

The analysis of spectroscopic data to solve chemical structures requires practical skills and drills. In this context, we have developed ULg Spectra, a computer-based tool designed to improve the ability of learners to perform complex reasoning. The identification of organic chemical compounds involves gathering and interpreting complementary information from mass, infrared, Raman, and nuclear magnetic resonance spectra. Here, special attention is paid to one-dimensional 1H and 13C NMR spectra and to two-dimensional NMR spectra because these techniques particularly require extensive interactive data manipulation. ULg Spectra offers tutorial-drill materials, including spectra that are “authentic” in the sense that they contain solvent and impurity traces rather than being “idealized” spectra often found in textbook examples. A public version is accessible online free of charge. The exam results for two groups of students, one having used ULg Spectra for extra home-based training and the other not, were com...

Aggregation Number in Water/n-Hexanol Molecular Clusters Formed in Cyclohexane at Different Water/n-Hexanol/Cyclohexane Compositions Calculated by Titration 1H NMR.

Upon titration of n-hexanol/cyclohexane mixtures of different molar compositions with water, water/n-hexanol clusters are formed in cyclohexane. Here, we develop a new method to estimate the water and n-hexanol aggregation numbers in the clusters that combines integration analysis in one-dimensional 1H NMR spectra, diffusion coefficients calculated by diffusion-ordered NMR spectroscopy, and further application of the Stokes-Einstein equation to calculate the hydrodynamic volume of the clusters. Aggregation numbers of 5-15 molecules of n-hexanol per cluster in the absence of water were observed in the whole range of n-hexanol/cyclohexane molar fractions studied. After saturation with water, aggregation numbers of 6-13 n-hexanol and 0.5-5 water molecules per cluster were found. O-H and O-O atom distances related to hydrogen bonds between donor/acceptor molecules were theoretically calculated using density functional theory. The results show that at low n-hexanol molar fractions, where a robust hydrogen-bond network is held between n-hexanol molecules, addition of water makes the intermolecular O-O atom distance shorter, reinforcing molecular association in the clusters, whereas at high n-hexanol molar fractions, where dipole-dipole interactions dominate, addition of water makes the intermolecular O-O atom distance longer, weakening the cluster structure. This correlates with experimental NMR results, which show an increase in the size and aggregation number in the clusters upon addition of water at low n-hexanol molar fractions, and a decrease of these magnitudes at high n-hexanol molar fractions. In addition, water produces an increase in the proton exchange rate between donor/acceptor molecules at all n-hexanol molar fractions.

Elucidation of 1H NMR Paramagnetic Features of Heterotrimetallic Lanthanide(III)/Manganese(III) 12-MC-4 Complexes.

The paramagnetic one-dimensional 1H NMR spectra of twelve LnIIINaI(OAc)4[12-MCMnIII(N)shi-4] complexes, where LnIII is PrIII-YbIII (except PmIII) and YIII, are reported. Their solid-state isostructural nature is confirmed in methanol-d4 solution, as a similar pattern in the 1H NMR spectra is observed along the series. Notably, a relatively well-resolved spectrum is reported for the GdIII complex. The chemical shift data are analyzed using the "all lanthanides" method, and the Fermi contact and pseudo-contact contributions are calculated for the lanthanide-induced shift (LIS). For the TbIII-YbIII complexes, the pseudo-contact contributions are typically 1 order of magnitude higher than the Fermi contact contributions; however, for the GdIII complex, the Fermi contact is the main contribution to the paramagnetic chemical shift. For the methyl protons of the axial acetate (-OAc) ligands, the LIS is opposite in sign, with respect to that of the aromatic salicylhydroximate (shi3-) protons, because of structural rearrangements that occur upon dissociation of the NaI cation in solution. The calculated crystal field parameters (BLn) for the TbIII (360 cm-1), DyIII (250 cm-1), HoIII (380 cm-1), ErIII (410 cm-1), TmIII (620 cm-1), and YbIII (380 cm-1) complexes are not constant, likely as a consequence of the inaccuracy of the Bleaney's constants and, to a smaller extent, the small structural changes that occur in solution. Overall, the metallacrown scaffold retains structural integrity and similarity in solution for the entire series; however, small structural features, which do not affect the overall similarity, do likely occur.

Hydrolysis of Tetraglycine by a Zr(IV)-Substituted Wells–Dawson Polyoxotungstate Studied by Diffusion Ordered NMR Spectroscopy

The use of diffusion ordered NMR spectroscopy (DOSY) for the analysis of complex reaction mixtures involving polyoxometalates (POMs) was demonstrated for the hydrolysis of the peptide tetraglycine by the K15H[Zr(α2-P2W17O61)2]·25H2O Wells–Dawson type cluster. 1H DOSY NMR studies have shown that severe signal overlap observed in the one-dimensional 1H NMR spectrum of reaction mixtures containing a POM and peptides could be overcome by the two-dimensional character of a DOSY NMR measurement. A clear distinction between the 1H NMR signals of the products formed during the hydrolysis of 5.0 mM of tetraglycine catalyzed by 1.0 mM of K15H[Zr(α2-P2W17O61)2]·25H2O was observed based on the extra dimension containing information about diffusion coefficients that distinguishes a typical DOSY measurement from conventionally used 1D 1H NMR. The spectrum clearly shows the presence of 5 species with diffusion coefficients of 3.71 × 10−10 m2/s (3.91; 3.84; 3.82 and 3.62 ppm), 4.39 × 10−10 m2/s (3.87; 3.76 and 3.61 ppm), 5.26 × 10−10 m2/s (3.67 and 3.63 ppm), and 7.46 × 10−10 m2/s (3.37 ppm) that are assigned to the non-hydrolyzed tetraglycine, the hydrolysis intermediate products triglycine and glycylglycine, and the end product of hydrolysis glycine, respectively. In addition, a signal assigned to cyclic glycylglycine, with a diffusion coefficient practically identical to the diffusion coefficient of glycylglycine was observed at 3.86 ppm. In addition, 1H and 31P NMR spectroscopy were further used to study the binding of tetraglycine to K15H[Zr(α2-P2W17O61)2]·25H2O and the solution speciation of K15H[Zr(α2-P2W17O61)2]·25H2O.

High-Resolution Quantitative Metabolome Analysis of Urine by Automated Flow Injection NMR

Metabolism is essential to understand human health. To characterize human metabolism, a high-resolution read-out of the metabolic status under various physiological conditions, either in health or disease, is needed. Metabolomics offers an unprecedented approach for generating system-specific biochemical definitions of a human phenotype through the capture of a variety of metabolites in a single measurement. The emergence of large cohorts in clinical studies increases the demand of technologies able to analyze a large number of measurements, in an automated fashion, in the most robust way. NMR is an established metabolomics tool for obtaining metabolic phenotypes. Here, we describe the analysis of NMR-based urinary profiles for metabolic studies, challenged to a large human study (3007 samples). This method includes the acquisition of nuclear Overhauser effect spectroscopy one-dimensional and J-resolved two-dimensional (J-Res-2D) 1H NMR spectra obtained on a 600 MHz spectrometer, equipped with a 120 μL flow probe, coupled to a flow-injection analysis system, in full automation under the control of a sampler manager. Samples were acquired at a throughput of ∼20 (or 40 when J-Res-2D is included) min/sample. The associated technical analysis error over the full series of analysis is 12%, which demonstrates the robustness of the method. With the aim to describe an overall metabolomics workflow, the quantification of 36 metabolites, mainly related to central carbon metabolism and gut microbial host cometabolism, was obtained, as well as multivariate data analysis of the full spectral profiles. The metabolic read-outs generated using our analytical workflow can therefore be considered for further pathway modeling and/or biological interpretation.

Combining Spectral Ordering with Peak Fitting for One-Dimensional NMR Quantitative Metabolomics

One-dimensional (1)H NMR spectra are widely used for metabolic profiling. Such data sets often contain hundreds or thousands of spectra, which typically have variation in their sample chemistry, which leads to chemical shift variation "positional noise". This is a severe problem for metabolite quantification and data analysis, as peak integrals do not necessarily correspond across all spectra in a set. Various alignment algorithms have been developed to address this problem, but different studies have taken different approaches to evaluating the performance of NMR alignment routines and can be subjective or rely on an arbitrary cutoff. Furthermore, most alignment approaches completely fail to deal with peaks that overlap. We adopt the simple and robust method of ordering spectra with respect to an internally varying peak and use this to compare different alignment algorithms. Furthermore, we use the information from this procedure to help improve a Bayesian approach to automated peak deconvolution by restricting the prior probability distribution of the peak position in a model-free manner and compare the performance to manual peak deconvolution and to binning. This combination of spectral ordering and compound deconvolution improved the quality of the data for quantitative metabolomics.

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

L-xylo-3-Hexulose Reductase Is the Missing Link in the Oxidoreductive Pathway for d-Galactose Catabolism in Filamentous Fungi

In addition to the well established Leloir pathway for the catabolism of d-galactose in fungi, the oxidoreductive pathway has been recently identified. In this oxidoreductive pathway, D-galactose is converted via a series of NADPH-dependent reductions and NAD(+)-dependent oxidations into D-fructose. The pathway intermediates include galactitol, L-xylo-3-hexulose, and d-sorbitol. This study identified the missing link in the pathway, the L-xylo-3-hexulose reductase that catalyzes the conversion of L-xylo-3-hexulose to D-sorbitol. In Trichoderma reesei (Hypocrea jecorina) and Aspergillus niger, we identified the genes lxr4 and xhrA, respectively, that encode the l-xylo-3-hexulose reductases. The deletion of these genes resulted in no growth on galactitol and in reduced growth on D-galactose. The LXR4 was heterologously expressed, and the purified protein showed high specificity for L-xylo-3-hexulose with a K(m) = 2.0 ± 0.5 mm and a V(max) = 5.5 ± 1.0 units/mg. We also confirmed that the product of the LXR4 reaction is D-sorbitol.

Functional Characterization of the FoxE Iron Oxidoreductase from the Photoferrotroph Rhodobacter ferrooxidans SW2

Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today.

Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p Paralogs Results in Catalytically Active Succinate Dehydrogenase Isoenzymes

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.

Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism

Histone H4 undergoes extensive post-translational modifications (PTMs) at its N-terminal tail. Many of these PTMs profoundly affect the on and off status of gene transcription. The molecular mechanism by which histone PTMs modulate genetic and epigenetic processes is not fully understood. In particular, how a PTM mark affects the presence and level of other histone modification marks needs to be addressed and is essential for better understanding the molecular basis of histone code hypothesis. To dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. Although certain acetylations present a repressive impact on PRMT1-mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases the activity of PRMT1 but increases that of PRMT5. Furthermore, circular dichroism study and computer simulation demonstrate that hyperacetylation increases the content of ordered secondary structures at the H4 tail region. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation and unravel the complex intercommunications that exist between different the PTM marks in cis. The divergent activities of PRMT1 and PRMT5 with respect to different acetyl-H4 substrates suggest that type I and type II protein-arginine methyltransferases use distinct molecular determinants for substrate recognition and catalysis.

TLR4 Recognizes Pseudallescheria boydii Conidia and Purified Rhamnomannans

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.

Using NMR Metabolomics to Investigate Tricarboxylic Acid Cycle-dependent Signal Transduction in Staphylococcus epidermidis

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to "sense" environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is "sensing" disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are "sensed" by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.