Biology subjects

Mentink-Vigier, F.

Publications and source records attributed to Mentink-Vigier, F..

8 recordsLinked to original sources

bioRxiv2026

Precursor-Dependent Routing of Aromatic Amino Acids Determines Lignin Structure in Grasses by Sensitivity-Enhanced Solid-State NMR

Lignin biosynthesis in grasses exhibits unique metabolic flexibility, yet the precursor-specific routing of carbon into lignin polymers remains poorly resolved in planta. Here, we combine 13C-isotope labeling with solid-state NMR under sensitivity-enhancement by dynamic nuclear polarization (DNP), to directly track phenylalanine- and tyrosine-derived carbon incorporation into the lignin polymer in Brachypodium distachyon. Precursor-specific 13C labeling reveals that phenylalanine is the dominant contributor to canonical guaiacyl and syringyl lignins, whereas tyrosine preferentially enriches hydroxyphenyl lignin and hydroxycinnamates, including ferulates characteristic of grass cell walls. Two-dimensional 13C-13C correlation NMR resolves distinct lignin moieties arising from each precursor. Disruption of p-coumarate 3-hydroxylase (C3H) selectively impairs phenylalanine-derived lignification, while tyrosine-derived lignin remains comparatively unchanged, maintaining polymer assembly through alternative metabolic routes. These findings show precursor-dependent control of lignin composition and reveal tyrosine-mediated lignification as a compensatory pathway in grasses. This work also establishes precursor-resolved solid-state NMR and DNP as a powerful framework for dissecting lignin biosynthesis and metabolic plasticity in plant cell walls. SIGNIFICANCE STATEMENTLignin is a complex plant polymer that strengthens cell walls but also limits the efficiency of biomass processing for agriculture and bioenergy. Grasses possess a unique lignin biosynthetic flexibility that is not well understood. By combining stable isotope labeling with solid-state NMR spectroscopy, we directly traced how the aromatic amino acids, phenylalanine and tyrosine, contribute differently to lignin formation in intact grass cell walls. We show that phenylalanine primarily builds conventional lignin structures, whereas tyrosine supplies alternative phenolic components and maintains lignin synthesis even when a key biosynthetic enzyme is disrupted. This metabolic flexibility helps explain the unique structural aspects of grass cell walls and identifies precursor-level control as a promising strategy for engineering lignin composition to improve biomass utilization.

plant biology↗

bioRxiv2025

Elucidation of Radical Degradation in Native Biofilms by EPR Sheds Light on Bacterial Resistance and Efficient DNP Solid-state NMR

Bacterial biofilms exhibit enhanced antimicrobial resistance, yet the mechanisms determining molecular transport and reactivity within these complex communities remain poorly understood. Here, we combine electron paramagnetic resonance (EPR), solid-state NMR (ssNMR), and dynamic nuclear polarization (DNP) ssNMR to determine how native Pseudomonas fluorescens Pf0-1 colony biofilms regulate nitroxide radicals. EPR measurements reveal that radical reduction depends on biofilm morphology, hydration, and composition of extracellular matrix (ECM). Wild-type biofilms exhibit slower nitroxide reduction than planktonic cells, while isolated ECM and dehydrated biofilms show no radical reduction, indicating that bacterial cells primarily drive radical reduction. Complementary ssNMR measurements identify polysaccharides and lipids within the ECM as primary interaction sites for nitroxide radicals. DNP-enhanced ssNMR further reveals compositional differences across biofilm morphologies, with increasingly polysaccharide-rich ECM environments correlating with slower reduction kinetics. These findings support a mechanism in which the ECM acts as a diffusion barrier and selective interaction region for nitroxide radicals to regulate cellular penetration. This work showcases an integrated magnetic resonance approach that provides molecular insight into how biofilm structure determines the fate of toxic redox-active small molecules. We also set the stage for high-sensitivity measurements of structure-function relationships in these medically relevant assemblies.

biophysics↗

bioRxiv2025

Polymorphic α-Glucans as Structural Scaffolds in Cryptococcus Cell Walls for Chitin, Capsule, and Melanin: Insights from 13C and 1H Solid-State NMR

Cryptococcus species are major fungal pathogens responsible for life-threatening infections in approximately a million individuals globally each year, with alarmingly high mortality rates. These fungi are distinguished by a distinctive cell wall architecture further reinforced by two virulence-associated layers, melanin and capsule, rendering them insensitive to antifungal agents targeting the cell wall, such as echinocandins. The molecular interplay between these three biomolecular layers remains poorly understood. Here we employ solid-state NMR spectroscopy to examine intact cells of both wild-type and capsule-deficient strains of C. neoformans, along with its melanized cells. High-resolution 13C and 1H data revealed five distinct structural forms of -1,3-glucans that play versatile roles in forming the rigid cell wall scaffold by interacting with chitin microfibrils and chitosan, and in stabilizing the mobile matrix by associating with {beta}-1,6-glucan and a small fraction of {beta}-1,3-glucan. Two primary forms of -1,3-glucans were distributed throughout the cell wall, hosting melanin deposition in the inner domain and capsule attachment on the cell surface. These findings offer a paradigm shift in understanding the cryptococcal cell wall and its interaction with two key virulence factors on opposite sides, raising critical biochemical questions that could inform the development of more effective antifungal treatments for cryptococcosis.

biochemistry↗

bioRxiv2024

Unveiling Cell Wall Structure and Echinocandin Response in Candida auris and Candida albicans via Solid-State NMR

Invasive candidiasis affects 1.6 million people annually, implicating high mortality and morbidity in immunocompromised and hospitalized patients. Echinocandins, inhibitors of {beta}-1,3-glucan synthesis, are used as a first-line treatment; however, their efficacy is increasingly compromised by resistance and tolerance. To understand how echinocandins remodel Candida cell wall structures, thereby reducing drug effectiveness, this study compares the effects of echinocandin exposure on the cell walls of the prevalent pathogen Candida albicans and the recently emerged multidrug-resistant superbug Candida auris. High-resolution solid-state NMR analysis revealed a conserved cell wall structure in both species, with a rigid inner layer composed of closely associated chitin microfibrils and {beta}-1,3-glucans, supported by a flexible network of {beta}-1,6-glucans and additional {beta}-1,3-glucans. Despite the presence of N-mannan fibrils in the outer layer, mannan components are mobile and rely on -1,2-linked mannoside sidechains to maintain contact with chitin and {beta}-1,3-glucans. Caspofungin treatment rigidifies certain mannan sidechains and {beta}-1, 6-glucans to reinforce the cell wall in response to the depletion of most {beta}-1,3-glucans. While caspofungin treatment reduced water permeability in both species, only C. albicans responded by inducing cell wall thickening and changes in chitin and {beta}-1,3-glucan dynamics. Furthermore, the deletion of KRE6 genes encoding {beta}-1,6-glucan synthase reduced the echinocandin susceptibility of C. auris, and the impaired {beta}-1,6-glucan biosynthesis were offset by compensatory upregulation of this wall component due to caspofungin treatment. The profound alterations induced by caspofungin in Candida cell wall architecture suggest that cell wall structural contribute substantially to drug resistance and tolerance.

microbiology↗

bioRxiv2024

High-Sensitivity Analysis of Native Bacterial Biofilms Using Dynamic Nuclear Polarization-Enhanced Solid-State NMR

Bacterial biofilms cause persistent infections that are difficult to treat and contribute greatly to antimicrobial resistance. However, high-resolution structural information on native bacterial biofilms remain very limited. This limitation is primarily due to methodological constraints associated with analyzing complex native samples. Although solid-state NMR (ssNMR) is a promising method in this regard, its conventional applications typically suffer from sensitivity limitations, particularly for unlabeled native samples. Through the use of Dynamic Nuclear Polarization (DNP), we applied sensitivity enhanced ssNMR to characterize native Pseudomonas fluorescens colony biofilms. The increased ssNMR sensitivity by DNP enabled ultrafast structural characterization of the biofilm samples without isotope-labelling, and chemical or physical modification. We collected 1D 13C and 15N, and 2D 1H-13C, 1H-15N and 13C-13C ssNMR spectra within seconds/minutes or hours, respectively which enabled us to identify biofilm components as polysaccharides, proteins, and eDNA effectively. This study represents the first application of ultrasensitive DNP ssNMR to characterize a native bacterial biofilm and expands the technical scope of ssNMR towards obtaining insights into the composition and structure of a wide array of in vitro and ex vivo biofilm applications. Such versatility should greatly boost efforts to develop structure-guided approaches for combating infections caused by biofilm-forming microbes.

microbiology↗

bioRxiv2024

Comparative Analysis of Polysaccharide and Cell Wall Structure in Aspergillus nidulans and Aspergillus fumigatus by Solid-State NMR

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13C, 15N, and 1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, -glucan, and {beta}-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.

biophysics↗

bioRxiv2023

Structural Remodeling of Fungal Cell Wall Promotes Resistance to Echinocandins

The insufficient efficacy of existing antifungal drugs and the rise in resistance necessitate the development of new therapeutic agents with novel functional mechanisms1,2. Echinocandins are an important class of antifungals that inhibit {beta}-1,3-glucan biosynthesis to interfere with cell wall structure and function3,4. However, their efficacy is limited by the fungistatic activity against Aspergillus species and the trailing effect during clinical application. Here, we describe how echinocandins remodel the supramolecular assembly of carbohydrate polymers in the fungal cell wall in an unexpected manner, possibly resulting in a subsequent inhibition of the activity of these drugs. Solid-state nuclear magnetic resonance (ssNMR) analysis of intact cells from the human pathogenic fungus Aspergillus fumigatus showed that the loss of {beta}-1,3-glucan and the increase of chitin content led to a decrease in cell wall mobility and water-permeability, thus enhancing resistance to environmental stresses. Chitosan and -1,3-glucan were found to be important buffering molecules whose physical association with chitin maintained the wall integrity. These new findings revealed the difficult-to-understand structural principles governing fungal pathogens response to echinocandins and opened new avenues for designing novel antifungal agents with improved efficacy.

biophysics↗

bioRxiv2023

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular environments

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition we find that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

biophysics↗