Plant cell walls contain multiple matrix polysaccharides whose interactions help determine wall mechanics, growth, and integrity, but it remains unclear how disruption of one biosynthetic or modification pathway reshapes other, non-target polymers. We used gel permeation chromatography coupled with enzyme-linked immunosorbent assay (GPC-ELISA) to compare mass distributions of major matrix polysaccharides in Arabidopsis thaliana mutants affecting xylan biosynthesis and decoration (irx14L, irx15, gux1-1, and gux2-2), glucomannan synthesis (csla2-1), pectin homogalacturonan (HG) biosynthesis (gaut10-2 and gaut11-2), and wall esterification (axy4-4, tbl31, tbl2, tbl3, and tbr). Three biological replicates per genotype, each from independent cell wall extractions, were profiled. Disruption of a single pathway frequently altered the distribution of non-target polymers. Xylan-related mutants altered not only glucuronoxylan but also HG and, in some cases, xyloglucan, with gux1-1 and gux2-2 producing particularly contrasting GX and HG profiles. By contrast, csla2-1 showed relatively limited secondary effects, whereas gaut10-2 and gaut11-2 showed increased low-molecular-size unesterified HG together with localised redistribution of several hemicellulose epitopes. Esterification mutants separated into distinct redistribution patterns rather than a single shared phenotype. Together, these findings support a directional and asymmetric model of cross-polymer coupling within extractable wall fractions, in which perturbation of HG or GX pathways has broader effects on the wall matrix than disruption of mannan biosynthesis.
Fucoidans, also known as fucose-containing sulfated polysaccharides (FCSPs), are abundant and structurally complex glycans found in the cell wall of brown algae. These compounds have been shown to have fundamental roles in the development and physiology of these organisms, to provide a major contribution to marine carbon sequestration, and to exhibit numerous bioactivities. Despite this, we currently have a limited understanding of the metabolic pathways and enzymes involved in synthesis and remodelling of fucoidans, their spatial and temporal dynamics in brown algae and in the marine environment, and how their structure and composition influence their bioactivities. Here, we highlight some of the challenges that have hindered progress on fucoidan research. We then moved on to underline some of the recent technical progress and new resources that would contribute to alleviate these challenges when combined together. This includes new technical approaches in analytical chemistry and glycobiology, as well the extension of genomic resources and genetics tools for brown algae. Finally, we suggest how progress in fucoidan research should contribute to advance understanding on brown algae biology, while supporting current and developing new aspects in fucoidan applications and seaweed biotechnology as part of the growing blue bioeconomy.
Mycolic acids are key components of the mycobacterial cell envelope, contributing to its structural integrity and intrinsic resistance to environmental stress. The polyketide synthase Pks13 catalyzes the final Claisen condensation step in mycolic acid biosynthesis and is conserved across Corynebacteriales. While the catalytic cysteine within the ketosynthase (KS) domain is established as essential for activity, the role of surrounding residues in supporting catalysis remains less well understood. Herein, we utilized a Mycobacterium smegmatis conditional pks13 mutant, and subsequent complemented strains harbouring a plasmid-borne copy of pks13 to probe the functional importance of selected residues within the KS domain from previous structural studies. These site-directed mutagenesis studies identified Asp264 as critical for Pks13 function. The D264A strain showed severely impaired growth along with a pronounced loss of mycolate synthesis and mycolate-containing lipids: trehalose dimycolate (TDM) and trehalose monomycolate (TMM). In contrast, other mutations had either little or no significant effect on cell viability or mycolate synthesis or mycolate-containing lipids. Structural modelling of the KS domain suggests that loss of Asp264 disrupts the local active-site environment, resulting in compaction of the binding pocket and altered conformation of a loop proximal to the catalytic residue Cys267. Together, these findings demonstrate that Asp264 is essential for Pks13 function and likely contributes to maintaining the structural environment of the KS domain.
The fungal pathogen Cryptococcus neoformans causes fatal cryptococcal meningitis, a significant global health risk. Unlike most fungi, C. neoformans expresses chitin deacetylases that convert its cell wall chitin into chitosan, helping to evade chitin-triggered host immunity. However, it is unclear how the properties and accessibility of these chitosans vary between strains and growth conditions, and how they influence the host immune response. To show how chitin deacetylase activity and growth conditions shape the properties of cell wall chitosans, we present the first comprehensive analysis of the chitosan content, fraction of acetylation (FA), enzymatic degradation products by host chitinases, and accessibility in various Cryptococcus strains, including chitin deacetylase mutants, cultivated under different conditions. Certain chitin deacetylase knockouts, as well as challenging growth conditions, lead to higher FAs and enhanced exposure, associated with stronger host immune responses. The response to cryptococcal chitin/chitosan ranges from low (resulting in cryptococcosis) to adequate (providing effective host defense) and excessive (leading to hyper-inflammation). Correlation between the strength of the immune response and the abundance of chitin-binding proteins was used to rapidly screen a multitude of strains and growth conditions for their immunogenic potential. Our approach provides insight into Cryptococcus-related disease caused by insufficient and excessive immune responses, and may accelerate the development of effective cell-based vaccines.
Efflux pumps confer antifungal resistance. Benzamidine (BM) conjugates overcome resistance by enhancing intracellular accumulation, but their cellular uptake mechanism remains unclear. A screen of membrane transport protein mutants revealed that plasma membrane transporters Alp1 and Nrt1 are importers of BM conjugates and are essential for their antifungal activity.
Cryptococcus neoformans is an important human fungal pathogen. Cell surface proteins mediate Cryptococcus-host interactions and play critical roles in fungal virulence. Proteins containing ricin B lectin-like domains (RBD) are involved in pathogen infection by interacting with exposed galactose residues of receptors on host cell surface. In this study, we screened the C. neoformans genome to identify proteins containing the ricin B lectin-like domain and identified five proteins that possess both a ricin B domain and a signal peptide. All five proteins contribute to cell wall integrity, particularly under high temperature. Four of these proteins localize to the cell wall and two are secreted to the culture medium. Expression of the RBD genes is positively regulated by the calcineurin-Crz1 pathway at host temperature. Furthermore, overexpression of either RBD2 or RBD5 restored the thermotolerance of the crz1Δ mutant to wild type levels. A comprehensive phenotypic analysis of these mutants revealed that Rbd proteins are involved in diverse functions, including the production of virulence factors, cell membrane and cell wall integrity, pathogen-host interactions, and fungal virulence in C. neoformans. This study enhances our understanding of the functions of Rbd proteins in fungal development and pathogenesis.
A comprehensive reference organism is often lacking when studying the adaptation of filamentous fungi to environmental stressors. This work investigates the mechanism underlying the response of N. crassa to stress conditions by focusing on the deletion mutant for the CCG-8 transcription factor. Our molecular analyses revealed that Δccg-8 severely compromises cell surface structure and metabolic homeostasis. Proteomic profiling demonstrated key dysregulations in ribosome biogenesis (consistent with the clock-controlled nature of CCG-8) and fatty acid β-oxidation. These findings, substantiated by changes in ergosterol and fatty acid composition, confirmed the increased susceptibility of deletion mutant to azoles and echinocandins. Furthermore, glycomic and proteomic data suggested that conidia of N. crassa Δccg-8 exhibit protein and glycan alterations. To validate this structural compromise in vivo, we successfully applied the Galleria mellonella larval model. Despite being non-pathogenic, conidia of N. crassa Δccg-8 were cleared significantly faster by the larval immune system than the wild-type strain, mirroring the in vitro observations. This work provides detailed molecular insights into fungal stress adaptation and establishes N. crassa as a viable non-pathogenic organism for in vivo analysis. This approach substantially broadens application of this filamentous fungus, enabling direct comparative research with pathogenic filamentous fungi in the domain of antifungal resistance and host interaction.
With the development of highly sensitive experimental techniques, the mechanical properties of bacterial cells have become an important research topic. However, existing models used to fit experimental data from micro-compression tests often lack accuracy. The aim of this study was to address this limitation by developing a new curve-fitting mathematical model for evaluating the mechanical properties of rod-shaped bacterial cells. The proposed model is based on a thin-shell approach and is specifically designed for the interpretation of single-cell micro-compression experiments.To verify the applicability of the model, single-cell micro-compression tests were performed using a flat-punch nanoindenter tip larger than the bacterial cells. Atomic force microscopy (AFM) was used to obtain detailed morphological information, including precise cell dimensions required for curve fitting. As a model organism, the polyhydroxyalkanoate-producing bacterium Cupriavidus necator H16 was selected due to its ability to accumulate intracellular polyhydroxybutyrate (PHB) granules. For comparison, a mutant strain, C. necator PHB−4, which lacks PHB production, was also analyzed.The results showed that C. necator H16 cells, with an average PHB content of 72% of dry cell weight, exhibited a Young's modulus approximately 16× higher than that of the PHB−4 mutant, indicating a substantial contribution of intracellular PHB granules to cell stiffness. AFM analysis further revealed that PHB-producing cells were, on average, larger in volume than the non-producing mutant. The combination of AFM and micro-compression testing enabled comprehensive characterization of bacterial cell mechanics and demonstrated a clear correlation between PHB content and mechanical behaviour.
Candida albicans is an opportunistic fungal pathogen that can cause a variety of superficial and life-threatening systemic infections. Relatively few clinically effective antifungal therapies are available, and the increasing prevalence of antifungal drug resistance poses a serious threat in treating these infections. Target validation of biochemical pathways that are essential for fungal growth offers an approach towards the design of novel antifungal drugs that address the growing requirement for new antifungal therapies. Therefore, we used the GRACE library of conditional mutants of C. albicans to explore enzymes in the sugar nucleotide biosynthesis pathway as potential drug targets. This pathway provides UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-glucose (UDP-Glc) and GDP-mannose (GDP-Man) substrates for the synthesis of the essential cell wall polymers, chitin, β-glucan(s) and mannan(s). We show that the genes encoding GDP-mannose pyrophosphorylase (SRB1/PSA1/VIG9), UTP-glucose-1-phosphaturidyl transferase (UGP1), phosphoglucose isomerase (PGI1) and glucosamine-6-phosphate synthase (GFA1) are critical for growth, biofilm formation and virulence in C. albicans. Genes encoding other enzymes in the sugar nucleotide biosynthetic pathway (namely AGM1, PMM1, PMI1, GNA1 and UAP1) were not essential for growth but were required for biofilm formation, tissue invasion and virulence. Repression of genes that encode these enzymes also resulted in hypersensitivity to a range of antifungal drugs as well as oxidative and cell wall stressors. These data underline the potential for augmenting antifungal drug development by targeting these enzymes in the treatment of C. albicans infections.
The fungicidal polyene amphotericin B (AMB) is the oldest antifungal for the treatment of systemic infections, and it remains a critical broad-spectrum therapeutic option, despite its well-documented nephrotoxicity. In many countries use of conventional amphotericin deoxycholate has been eclipsed by the introduction in the 1990s of the considerably more expensive but much less nephrotoxic lipid formulations. Amphotericin B is valued for its strong fungicidal activity at low doses and its rarity of resistance, as resistance usually carries a significant fitness cost for fungi. However, emerging pathogens such as Candidozyma auris (formerly Candida auris) often exhibit significant resistance levels, with >30 % of clinical isolates showing reduced AMB susceptibility. Like azoles, AMB targets ergosterol in the fungal membrane, but unlike azoles, it binds pre-existing ergosterol; however, reduced ergosterol alone does not fully explain emerging resistance. Recent studies have revealed novel, often sterol-independent mechanisms related to sphingolipid content that drive AMB resistance, particularly in yeast species. These findings broaden our understanding and emphasize critical knowledge gaps. We review these evolving mechanisms and the pressing need for further research into the evolution of AMB resistance pathways.
Schizophyllan, a β-(1,3)(1,6)-glucan, is part of the cell wall of the mushroom-forming fungus Schizophyllum commune and is also released into the culture medium. It has various commercial applications but the natural function of schizophyllan during growth of S. commune is largely unknown. The S. commune strain H4-8A was grown on minimal medium (MM-N) and medium containing 10-fold more KH2PO4/K2HPO4 buffer (MM-NKP). The addition of extra buffer resulted in a 4.2-fold decrease in water-soluble schizophyllan and a 8.8-fold decrease in rigid schizophyllan in the cell wall. This decrease in schizophyllan was associated with a 3.7 fold lower tensile strength and a 2.5-fold higher elasticity of the mycelium. Moreover, spores of S. commune, as well as cells of Escherichia coli and Pseudomonas putida, showed increased survival against heat treatment and freeze-thawing, and had a longer shelf-life in the presence of schizophyllan. Also, schizophyllan can be metabolized by S. commune but the tested bacteria were unable to do so. Together, schizophyllan provides rigidity to the cell wall, protects S. commune against temperature stress, and can be used as an external carbon storage. It may also form a selective barrier around the hyphae, protecting S. commune against attack by bacteria.
Sporothrix schenckii and Sporothrix brasiliensis are causative agents of sporotrichosis, a mycosis that affects humans and other mammals. Adhesins are considered among the primary virulence factors of these species; however, their molecular identities are currently scarce. Here, we generated silenced mutant strains in PAP1, encoding a peptidorhamnomannan-associated protein, in both fungal species and compared their phenotypical characterization. PAP1-silenced mutants had normal growth, dimorphism, and morphology, suggesting the gene is not essential. The silenced strains showed defects in adhesion to HeLa cells, laminin, fibronectin, fibrinogen, type-I collagen, and type-II collagen. The S. schenckii silenced mutants showed a significant reduction in the adhesion to elastin, whilst the S. brasiliensis mutant showed impaired ability to bind thrombospondin 1 and to form biofilms. Both sets of PAP1-silenced mutants displayed defects in the cell wall composition, with reduced levels of cell wall rhamnose. The S. schenckii mutants showed a compensatory mechanism increasing cell wall mannose content, while the S. brasiliensis mutants increased cell wall protein levels in compensation to PAP1 silencing. Both mutant sets changed their ability to stimulate cytokine production in human peripheral blood mononuclear cells and monocyte-derived macrophages. In addition, phagocytosis of S. schenckii PAP1-silenced strains was significantly increased, while the S. brasiliensis PAP1-silenced strains were poorly phagocytosed. Finally, PAP1 silencing affected virulence in both species.These results suggest that S. schenckii and S. brasiliensis PAP1 code for a protein with adhesive properties to different ligands. This is a cell wall protein that contributes to Sporothrix virulence and the interaction with immune cells.
Fosmanogepix is a new antifungal agent currently undergoing phase 3 clinical trials. Its active moiety, manogepix, inhibits Gwt1 which is an enzyme essential for the assembly and attachment of glycosylphosphatidylinositol anchors to cell wall proteins. Manogepix has a strong fungistatic activity against most human pathogenic fungal species. Here we characterized the activity of manogepix against the major human pathogenic mold Aspergillus fumigatus. We show that manogepix susceptibility is linked to the expression of gwt1, the gene encoding Gwt1, thereby demonstrating that overexpression of gwt1 can confer resistance. In agreement with a previous study conducted with a different fungal pathogen, we observed an increase of cell well chitin after manogepix treatment. Using a luciferase-based reporter assay, we show that manogepix activates the cell wall integrity stress response pathway. However, manogepix only occasionally causes cell lysis. Mutants that lack the cell wall stress sensor Wsc1 or the Rho GTPase Rho4, which is important for septum formation, are slightly more susceptible to manogepix. In contrast, mutants lacking the Rho GTPase Rho2 or, to a lesser extent, the cell wall stress sensor MidA, both of which are essential for A. fumigatus to survive attacks from granulocytes and other forms of cell wall stress, grow better than wild-type when exposed to manogepix. We show that both mutants, especially Δrho2 but also ΔmidA, fail to upregulate cell wall chitin to wild-type-like levels in response to manogepix. These results implicate a role for the Rho2-dependent stress response in the fungistatic activity of manogepix against A. fumigatus.
The cell wall plays a central role in maintaining bacterial shape and structural stability. In the Corynebacteriaceae family, which includes Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus, the envelope has a distinctive organization: peptidoglycan is covalently attached to arabinogalactan, which is subsequently enveloped by an outer layer of mycolic acids. In this study, we used Corynebacterium glutamicum as a model to examine how disruptions in different layers of the cell envelope, specifically the arabinogalactan and mycolic acid layers, influence both biochemical and biophysical properties. We assessed surface hydrophobicity, antibiotic sensitivity, and lipid composition, and complemented these assays with atomic force microscopy to examine structural changes. Our analysis of multiple cell wall mutants suggests that a division of labour occurs among the layers of the corynebacterial envelope, where each layer contributes to distinct, but complementary functions to overall cell wall physiology.
Murein lipoprotein (Lpp), also known as Braun's lipoprotein, stabilizes the cell wall of Escherichia coli by covalently tethering the outer membrane to the peptidoglycan (PG). Unlike E. coli, Salmonella enterica serovar Typhimurium encodes two murein lipoproteins, LppA and LppB, with LppB bearing an unusual C-terminal sequence, -RICKCOOH. Here, we investigated how LppA and LppB bind to the PG. Both lipoproteins were detected in pure PG material in a ∼ 400:1 LppA:LppB ratio with some LppB molecules forming a cysteine 78 (C78)-C78 intermolecular disulphide bridge. LppA and LppB anchor covalently to uncross-linked and cross-linked muropeptides. However, unlike LppA, which binds to 4,3- and 3,3-cross-linked muropeptides, LppB shows preferred binding to 4,3-cross-linked muropeptides. Mass spectrometry data revealed O-methylation at the terminal K79 residue in some PG-bound LppB molecules. The apparent selective anchoring of LppB to the PG and the K79 modification require the presence of the C78 residue. Anchoring of LppB to PG is mediated by the L,D-transpeptidase LdtB. A survey in more than 158,000 Salmonella genomes identified up to 31 murein Lpp variants differing in the C-terminal region that cluster in three phylogenetic groups. Most serovars of S. enterica subspecies enterica, responsible for infections in warm blooded animals, encode two or even three murein Lpp variants. Altogether, our data are consistent with subtle differences in the mode that LppB anchors to the PG and uncover an unprecedented diversity of murein lipoproteins within the Salmonella genus. The possibility that this variability evolved as strategy to evade host innate immunity, is also discussed.
Bacterial infections trigger robust host immune responses, while pathogens concurrently adapt to enhance survival within the host. In our previous study, we observed 24 h of Salmonella Typhi infection in Caenorhabditis elegans colonized the body of the host and also vertically transmitted to their F1 offspring generation. This study investigates host interaction-mediated modulation in S. Typhi physiology and virulence, focusing on host-derived strains from initial infection (CeP0-ST) and vertically transmitted F1 progeny (CeF1-ST), compared to the wild-type unexposed strain (WT-ST). We employed C. elegans lifespan assays, bacterial colonization, morphology, motility, gene expression, ROS estimation, and infection models using immune pathway mutants. CeP0-ST and CeF1-ST exhibited increased infectivity, faster mortality, and enhanced colonization, accompanied by reduced cell size, motility, and lipopolysaccharide (LPS)-mediated immunogenicity. This was evidenced by downregulation of fliC, ompC, ompF, and SPI-1 encoded Type III Secretion System (T3SS-1) genes (sipA, avrA, sopE). Upregulation of Vi-capsular antigen genes (tviD, tviA, viaB) suggested immune evasion, which was supported by improved host survival upon Vi-negative Ty21a strain infection. Among various C. elegans immune pathway mutants, the TGF-β pathway mutant [sma-6(-)] showed altered pathogen modulation, with reduced Vi expression and increased T3SS gene expression in derived strains. Suppressed host immune gene expression during WT-derived infections, but increased expression during sma-6 (-)-derived infections, suggests a role for TGF-β signalling in driving pathogen adaptation. These findings highlight that host interaction promotes S. Typhi immune evasion via Vi-antigen expression, potentially regulated by host TGF-β signalling, and that vertical transmission of adapted strains facilitates long-term persistence.
Sporothrix brasiliensis and Sporothrix schenckii are major causative agents of sporotrichosis, a neglected fungal disease with growing public health relevance. These pathogens release extracellular vesicles (EVs) that contribute to host-pathogen interactions, yet their small molecule content remains unexplored. Here, we isolated EVs from both species following growth on solid medium and characterized them using nanoparticle tracking analysis, transmission electron microscopy, proteomics, and untargeted metabolomics. While EVs from each species contained distinct sets of proteins, a subset of conserved components and metabolites was identified, including the dipeptidyl peptidase IV (DPP4) inhibitor isoleucine-proline-isoleucine (IPI), previously described as a protective EV component in Cryptococcus. Functional assays demonstrated that both IPI and an anti-DPP4 antibody significantly impaired fungal adhesion to type I collagen. These findings reveal new layers of molecular complexity in the EVs of Sporothrix spp., highlight the presence of functionally conserved small molecules, and suggest a role for EV-derived peptides in modulating fungal surface interactions with host structures.
Characterization of microalgae cell wall properties using Atomic Force Microscopy (AFM) depends critically on the immobilization strategy employed. In this study, we investigated how various immobilization surfaces influence the biophysical properties of the Chlorella vulgaris cell wall as probed by AFM. Cells were immobilized on three distinct surfaces, each leveraging different types of interactions with cells: polyoctyl-(PO)-Chitosan-coated glass slides promoting hydrophobic interactions, Superfrost™ positively charged glass slides that interact hydrophobically and electrostatically with cells, and PDMS chambers that immobilize cells through mechanical confinement. AFM imaging, nanoindentation, and force spectroscopy revealed that the biophysical properties of the Chlorella vulgaris cell wall strongly depend on the immobilization strategy. At the same time, all other experimental conditions were kept identical. Cells attached to PO-Chitosan through hydrophobic interactions displayed high surface roughness, higher rigidity, hydrophobic behavior, and positive surface charges. In contrast, cells immobilized on positively charged but less hydrophobic Superfrost™ slides exhibited smoother surfaces, lower rigidity, reduced hydrophobicity, and no detectable positive charges. Cells confined in PDMS chambers exhibited the smoothest and softest cell walls, characterized by hydrophilic and uncharged surfaces. Complementary Stimulated Raman Scattering (SRS) microscopy experiments confirmed distinct lipid rearrangements associated with each immobilization method. Together, these results support the hypothesis formulated in this study that immobilization surfaces can cause the reorganization of cell wall components—polysaccharides, proteins, and lipids—thereby reshaping the physico-chemical and mechanical surface properties. This study thus demonstrates the great significance of immobilization strategies to ensure the reliability of nanoscale biophysical measurements in microalgae research.
Plant and fungal cell walls are essential for growth, adaptation, and survival, with their intricate architectures dictating both resistance to stress and susceptibility to antifungal or biomass-degrading strategies. Understanding how these walls are built, remodeled, and function at the molecular level is therefore central to both clinical and biotechnological applications. Solid-state nuclear magnetic resonance (ssNMR) has emerged as a uniquely powerful tool for this purpose, as it reveals the structure, dynamics, and interactions of intact biopolymers without disrupting their native organization. Using this approach, recent studies have shown how structural polymorphism, polymer-polymer interactions, and species-specific remodeling govern mechanical integrity, drug resistance, and stress adaptation. Applications highlighted here include lignin-carbohydrate packing during plant stem maturation, fungal wall reorganization under treatment by wall-targeting antifungals such as echinocandin and nikkomycin, and the functional diversity of glucans, chitins, and mannans. Together, these insights uncover conserved principles of polymer assembly across kingdoms while informing new opportunities for antifungal development and biomass utilization. Ongoing advances in sensitivity and resolution are expected to broaden the reach of ssNMR and further accelerate its role in linking structural heterogeneity to biosynthetic complexity and biological function.
