Cell Wall Layers Research Articles

Fungi hijack a ubiquitous plant apoplastic endoglucanase to release a ROS scavenging β-glucan decasaccharide to subvert immune responses.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of β-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant–microbe interface.

XET activity determination in powdered wood samples as an indicator of tension wood, tested on juvenile Populus x euramericana exposed to severe long-term static bending

Abstract Leaning stems of woody plants form reaction wood, in hardwood trees termed tension wood (TW). Typical TW fibers, gelatinous fibers (G-fibers), are characterized by an inner gelatinous cell wall layer (G-layer). Xyloglucan endotransglycosylases (XETs) was proposed as the essential enzyme in cell wall modifications in TW, by making xyloglucan (XG) cross-links between G- and S2-layers in G-fibers, and thus maintaining their contact. The determination of TW presence in a sample is of great importance for the forest products industry, biofuel production, and tree physiology studies. However, TW is not easy to detect visually. The colorimetric assay for XET activity determination as an indicator of TW presence in a sample was tested on powdered stem segments of juvenile Populus x euramericana trees exposed to severe long-term static bending. In parallel, histochemical and ultrastructural characterization of stem samples of bent and control trees was performed. The tested colorimetric assay for XET activity determination could be suggested as a useful and easily applicable tool for fast screening of powdered wood samples for the presence of TW.

In situ microstructural evolution of spruce wood during soda pulping using synchrotron X-ray tomography

Abstract A new laboratory reactor to perform in situ studies of structural changes in wood during soda pulping using synchrotron X-ray tomography is presented. The reactor is of recirculation type to provide stable reaction conditions and mimic the industrial situation. Experiments have been performed using this reactor in situ at a synchrotron microtomography beamline to provide sequences of 3D images from which measurement of wood cell wall thickness have been possible for the first time. The results showed that the cell wall thickness increased significantly in the early stage of pulping (<10 min), which is due to the transportation of cooking chemicals through the tracheids, resin channels and pits into the cell wall, which is swollen with the increased pH. Subsequently, the cell wall thickness reduces over the processing time up to 60 min with a high rate, which is inferred to be due to the dissolution and transport of lignin and hemicellulose from the secondary walls, allowing for better transportation of active chemicals deep through the cell wall layers. After 60 min processing, the cell wall thickness reduction rate reduced, as dissolution of lignin and hemicelluloses from the cell walls ceased, while the remaining dissolution occurs mainly at the middle lamella.

Chemical Characteristics of Wood Cell Wall with an Emphasis on Ultrastructure: A Mini-Review

Wood is complex in its chemical composition that has an important influence on its chemical behavior and mechanical strength. The complexity is reflected in the ultrastructure of the wood cell wall. In particular, the concentration of main components (cellulose, hemicelluloses and lignin) changes depending on many factors such as the different type or parts of wood, and varies in different cell wall layers. From an ultrastructural standpoint, we describe the current level of knowledge about chemical characteristics of the wood cell walls. The information of distribution of main components in the cell walls of normal wood, reaction wood and water-logged archaeological wood, the cellulose microfibrils orientation, and the interactions between main components were presented based on the use of advanced techniques including transmission electron microscopy, scanning electron microscopy, spectral imaging and nuclear magnetic resonance. In addition, the chemical changes of the wood cell wall during pretreatment are discussed. This mini-review not only provides a better understanding of wood chemistry, but also brings new insights into cell wall recalcitrance.

Halorubrum pleomorphic virus-6 Membrane Fusion Is Triggered by an S-Layer Component of Its Haloarchaeal Host.

(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6.

Bacterial cell wall material properties determine E. coli resistance to sonolysis

The applications of bacterial sonolysis in industrial settings are plagued by the lack of the knowledge of the exact mechanism of action of sonication on bacterial cells, variable effectiveness of cavitation on bacteria, and inconsistent data of its efficiency. In this study we have systematically changed material properties of E. coli cells to probe the effect of different cell wall layers on bacterial resistance to ultrasonic irradiation (20 kHz, output power 6,73 W, horn type, 3 mm probe tip diameter, 1 ml sample volume). We have determined the rates of sonolysis decay for bacteria with compromised major capsular polymers, disrupted outer membrane, compromised peptidoglycan layer, spheroplasts, giant spheroplasts, and in bacteria with different cell physiology. The non-growing bacteria were 5-fold more resistant to sonolysis than growing bacteria. The most important bacterial cell wall structure that determined the outcome during sonication was peptidoglycan. If peptidoglycan was remodelled, weakened, or absent the cavitation was very efficient. Cells with removed peptidoglycan had sonolysis resistance equal to lipid vesicles and were extremely sensitive to sonolysis. The results suggest that bacterial physiological state as well as cell wall architecture are major determinants that influence the outcome of bacterial sonolysis.

Molecular and Microscopic Methods of Quantifying Candida albicans Cell Wall PAMP Exposure.

The cell wall of Candida albicans is a multilayered structure consisting of polysaccharides and proteins. The inner cell wall layer is comprised of chitin and β1-3 and β1-6-glucan which contribute to the overall shape and structure of the cell, while the outer layer of highly glycosylated mannoproteins provides key functional traits such as cell adhesion required for virulence. However, the cell wall is not a static structure but is constantly being remodeled in response to the external environment. Given that all of the cell wall components act as pathogen-associated molecular patterns (PAMPs) that are recognized by a variety of receptors on the surface of innate immune cells, remodeling of the cell wall can have a dramatic impact on the host-pathogen interaction. For example, during growth in standard media, C. albicans shields its major cell wall PAMPs from the innate immune system, but during growth in acidic environments as encountered during colonization of the female reproductive tract, key PAMPs become exposed on the fungal cell surface initiating a strong pro-inflammatory innate immune response. The impact of environmental adaptation on fungal cell wall remodeling, and the subsequent impact this has on the host-pathogen interaction, has been the subject of much research. In this chapter, we outline techniques to assess cell wall components in both resting and environmentally adapted C. albicans cells.

Cytological Observation of the Infectious Process of Venturia carpophila on Peach Leaves

Peach scab caused by Venturia carpophila is one of the most destructive fungal diseases of peach worldwide, and it seriously affects peach production. Until now,the infectious process and pathogenesis of V. carpophila on peach have remained unclear. Here we present the infection behavior of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (i.e., light microscopy, confocal laser scanning microscopy, scanning electron microscopy, and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer and then differentiated into thick subcuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the subcuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the subcuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the subcuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further a understanding of V. carpophila pathogenesis.

Effective solvents for proteins recovery from microalgae

From an industrial perspective, the exploitation of microalgae as protein source is of great economical and commercial interest due to several attractive characteristics. However, the protein extraction efficiency is limited by the multiple layers of rigid and thick cell walls that are enriched with recalcitrant structure of cellulose. Therefore, an efficient method of cell disruption is necessary to disintegrate the cell wall and promote the release of protein contents. The conventional methods for downstream processing, e.g. disruption, isolation, extraction, concentration and purification, are energy-intensive and costly because they typically compose of several operational units. To reduce the overall process cost and establish an economical feasible process for the large-scale production of microalgae derived products, a more cost-effective and ecofriendly technique in downstream processing is in critical demand. One of the main challenges for protein extraction from microalgae cells is the recalcitrant structure of microalgae cell wall. This work aims to provide a guideline on the selection of the solvent to facilitate the proteins release during the cell disruption process. The influences of various solvent types (methanol, ethanol, 1-propanol and water) were evaluated and compared based on the protein yields. It was found that water solvent released the highest protein concentration from microalgae compared to the other tested solvents.

Therapeutic regimens of endotoxaemia in sheep

Endotoxaemia is an inflammatory condition which happens due to the presence of outer cell wall layer of Gram-negative bacteria in blood circulation, containing lipopolysaccharide commonly known as endotoxin. This condition causes high mortality in affected animals and sheep are highly susceptible in this regard. Several researchers have emphasised the therapeutic regimens of endotoxaemia and its sequels in sheep. Furthermore, sheep are among the most commonly used animal species in experimental studies on endotoxaemia, and for the past five decades, ovine models have been employed to evaluate different aspects of endotoxaemia. Currently, there are several studies on experimentally induced endotoxaemia in sheep, and information regarding novel therapeutic protocols in this species contributes to better understanding and treating the condition. This review aims to specifically introduce various treatment methods of endotoxaemia in sheep.

Evolution and properties of alanine racemase from Synechocystis sp. PCC6803.

Alanine racemase (EC 5.1.1.1) depends on pyridoxal 5'-phosphate and catalyses the interconversion between L- and D-Ala. The enzyme is responsible for the biosynthesis of D-Ala, which is an essential component of the peptidoglycan layer of bacterial cell walls. Phylogenetic analysis of alanine racemases demonstrated that the cyanobacterial enzyme diverged before the separation of gram-positive and gram-negative enzymes. This result is interesting considering that the peptidoglycans observed in cyanobacteria seem to combine the properties of those in both gram-negative and gram-positive bacteria. We cloned the putative alanine racemase gene (slr0823) of Synechocystis sp. PCC6803 in Escherichia coli cells, expressed and purified the enzyme protein and studied its enzymological properties. The enzymatic properties of the Synechocystis enzyme were similar to those of other gram-positive and gram-negative bacterial enzymes. Alignment of the amino acid sequences of alanine racemase enzymes revealed that the conserved tyrosine residue in the active centre of most of the gram-positive and gram-negative bacterial enzymes has been replaced with tryptophan in most of the cyanobacterial enzymes. We carried out the site-directed mutagenesis involving the corresponding residue of Synechocystis enzyme (W385) and revealed that the residue is involved in the substrate recognition by the enzyme.

Subtractive genomic approach toward introduction of novel immunogenic targets against Clostridioides difficile: Thinking out of the box

Clostridioides difficile is one of the major causatives of nosocomial infections worldwide. Antibiotic-associated diarrhea, pseudomembranous colitis, and toxic megacolon are the most common forms of C. difficile infection (CDI). Considering the high antibiotic resistance of C. difficile isolates and the low efficacy of immunization with toxin-related vaccines, we suggested that surface-exposed and secreted proteins could be considered as potential immunogenic targets against CDI. Various immuninformatics databases were used to predict antigenicity, allergenicity, B-cell epitopes, MHC-II binding sites, conserved domains, prevalence and conservation of proteins among the most common sequence types, molecular docking, and immunosimulation of immunogenic targets. Finally, 16 proteins belonging to three functional groups were identified, including proteins involved in the cell wall and peptidoglycan layer (nine proteins), flagellar assembly (five proteins), spore germination (one protein), and a protein with unknown function. Molecular docking results showed that among all the mentioned proteins, WP_009892971.1 (Acd) and WP_009890599.1 (a C40 family peptidase) had the strongest interactions with human Toll-like receptor 2 (TLR-2) and TLR-4. This study proposes a combination of C. difficile toxoid (Tcd) and surface-exposed proteins such as Acd as a promising vaccine formulation for protection against circulating clinical strains of C. difficile.

Spatial mapping of immunological epitopes in the Candida cell wall using C-type lectin probes

The primary recognition event between a fungal pathogen and the immune system normally involves the engagement of a pattern recognition receptor with specific components of the cell wall. However, the cell wall is a complex three dimensional structure whose composition changes rapidly in accordance with environmental stimuli. Therefore it is important to know what is the precise nature of the primary recognition event, how many events occur to activate the immune response and how these recognition events are affected by changes in cell wall architecture, cellular morphogenesis and physiological adaptation of the pathogen to specific niches in the human body. We address this fundamental question using four soluble immune C-Type lectin receptor-probes which recognize specific mannans and β-1,3 glucan in the cell wall. We use these C-type lectin probes to demonstrate that mannan epitopes are differentially distributed in the inner and outer layers of fungal cell wall in a clustered or diffuse manner. Immune reactivity of fungal cell surfaces did not correlate with relatedness of different fungal species, and mannan-detecting receptor-probes discriminated between cell surface mannans generated by the same fungus growing under different conditions. These studies demonstrate that mannan-epitopes within fungal cell walls are differentially distributed and dynamically expressed as the fungus adapted to microenvironments that would be encountered in vivo.

Insight into the negative effects of lignin on enzymatic hydrolysis of cellulose for biofuel production via selective oxidative delignification and inhibitive actions of phenolic model compounds

Oxidative pretreatment of wheat straw with sodium chloride has been used for selective delignification to obtain ideal samples to understand the negative effects of lignin on cellulose accessibility of lignocellulosic biomass. Strong interactive effects has been found between hemicellulose and lignin. To achieve a high enzymatic glucan conversion (EGC) (>80% with 20 FPU/g solid) in the case of high xylan content (25–30%), a moderate degree of delignification (56.3%) with relatively low lignin content (11.9%) is necessary; while in the case of low xylan content (2.6–3.9%), slight removal of lignin by oxidative treatment can well increase cellulose digestibility with a final EGC reaching 90% even that the residual lignin content is as high as 30.9%. Delignification greatly modifies substrate surface morphology with deformation, fracture of cell wall layers and even disappearance of middle lamella. Oxidation also modifies the functional groups and surface properties of lignin, leading to reduction in the inhibitive effects. Phenolic hydroxyl group (Ph-OH) in p-hydroxyphenyl unit has been found to show the strongest inhibition to filter paper activity of cellulase cocktail. Ph-OH exerts stronger inhibition to β-glucosidase than to the endo- and exo-glucanases. Hydrogen bonding and electrostatic interaction are primarily responsible for the inhibitive action of Ph-OH.

Best Practices for Quasistatic Berkovich Nanoindentation of Wood Cell Walls

For wood and forest products to reach their full potential as structural materials, experimental techniques are needed to measure mechanical properties across all length scales. Nanoindentation is uniquely suited to probe in situ mechanical properties of micrometer-scale features in forest products, such as individual wood cell wall layers and adhesive bondlines. However, wood science researchers most commonly employ traditional nanoindentation methods that were originally developed for testing hard, inorganic materials, such as metals and ceramics. These traditional methods assume that the tested specimen is rigidly supported, homogeneous, and semi-infinite. Large systematic errors may affect the results when these traditional methods are used to test complex polymeric materials, such as wood cell walls. Wood cell walls have a small, finite size, and nanoindentations can be affected by nearby edges. Wood cell walls are also not rigidly supported, and the cellular structure can flex under loading. Additionally, wood cell walls are softer and more prone to surface detection errors than harder inorganic materials. In this paper, nanoindentation methods for performing quasistatic Berkovich nanoindentations, the most commonly applied nanoindentation technique in forest products research, are presented specifically for making more accurate nanoindentation measurements in materials such as wood cell walls. The improved protocols employ multiload nanoindentations and an analysis algorithm to correct and detect errors associated with surface detection errors and structural compliances arising from edges and specimen-scale flexing. The algorithm also diagnoses other potential issues arising from dirty probes, nanoindenter performance or calibration issues, and displacement drift. The efficacy of the methods was demonstrated using nanoindentations in loblolly pine (Pinus taeda) S2 cell wall layers (S2) and compound corner middle lamellae (CCML). The nanoindentations spanned a large range of sizes. The results also provide new guidelines about the minimum size of nanoindentations needed to make reliable nanoindentation measurements in S2 and CCML.

Paeonol Disrupts the Integrity of Aspergillus flavus Cell Walls via Releasing Surface Proteins, Inhibiting the Biosynthesis of β-1,3-Glucan and Promoting the Degradation of Chitin, and an Identification of Cell Surface Proteins.

Paeonol can effectively inhibit Aspergillus flavus (A. flavus) via damaging cell walls. In this work, paeonol treatment remarkably destroyed both the outer amorphous layer and the inner fibrous layer of cell walls. Furthermore, FT-IR and XPS characterization showed that OH functional groups were altered and proteins in the outer layer were released. According to proteomic analysis, 605 proteins have been identified and annotated. The activities of β-1,3-glucan synthase and chitinase were prohibited and promoted, respectively, by paeonol treatment, however, the activities of β-1,3-glucanase and chitin synthase were not influenced. QRT-PCR results suggested that FKSP, CHIIII, and CHIV genes might be the antifungal targets of paeonol. In addition, paeonol can effectively restrain the pathogenicity of A. flavus on peanut butter. This study provided a new elucidation on the mode of action of paeonol against cell walls of A. flavus, facilitating the application of paeonol in the preservation of agricultural products.

Local force titration of wood surfaces by chemical force microscopy

Chemical force microcopy, a variation of atomic force microscopy, opened the door to visualize chemical nano-properties of various materials in their natural state. The key function of this method is given by translating adhesion forces between a functionalized tip and the sample to chemical surface behavior. In force titration, these adhesion forces are studied in different pH buffers, which allows estimating the pKa value of the analyzed surface. Herein, we report the use of this method to study natural and chemically treated wood surfaces, which are of interest in sustainable material design. First, we show varying adhesion phenomena of OH- and COOH-functionalized tips on native spruce wood cells. Then, we demonstrate how peak force tapping with chemically functionalized tips can be used to estimate the pKa value of gold substrates (pKa ≈ 5.2) and different wood cell wall layers with high spatial resolution. Additionally, the swelling behavior of wood samples is analyzed in varying pH buffers. With the applied method, chemical surface properties of complex natural substrates can be analyzed.Graphical abstract

Dislocation cell walls with high dislocation density as effective hydrogen traps in Armco iron

AbstractThe effect of cold rolling (CR) deformation on the hydrogen permeation and trapping behavior of pure iron was systematically investigated. Increased deformation resulted in a decreased effective hydrogen diffusion coefficient, an extended effective lattice interstitial hydrogen diffusion path length, and increased hydrogen trap densities. However, the proportion of irreversible hydrogen traps to all traps was constant irrespective of the deformation level in the range of 30%–70% CR and increased when the deformation reached 80%. This is because the dislocation cell wall is composed of a central layer with high trap‐binding capacity and an outer layer with low trap‐binding capacity. More importantly, once the dislocation density in the center layer of the dislocation cell walls reached a relatively high value due to the severe plastic deformation, these regions become effective irreversible hydrogen traps.

Bordered Pit Formation in Cell Walls of Spruce Tracheids.

The process of pit formation in plants still has various questions unaddressed and unknown, which opens up many interesting and new research opportunities. The aim of this work was elucidation of the mechanism for the formation of bordered pits of the spruce (Picea abies (L.) Karst.) tracheid with exosomes participation and mechanical deformation of the cell wall. Sample sections were prepared from spruce stem samples after cryomechanical destruction with liquid nitrogen. The study methods included scanning electron microscopy and enzymatic treatment. Enzymatic treatment of the elements of the bordered pit made it possible to clarify the localization of cellulose and pectin. SEM images of intermediate stages of bordered pit formation in the radial and tangential directions were obtained. An asynchronous mechanism of formation of bordered-pit pairs in tracheids is proposed. The formation of the pit pair begins from the side of the initiator cell and is associated with enzymatic hydrolysis of the secondary cell wall and subsequent mechanical deformation of the primary cell walls. Enzymatic hydrolysis of the S1 layer of the secondary cell wall is carried out by exosome-delivered endoglucanases.

Defining Functions of Mannoproteins in Saccharomyces cerevisiae by High-Dimensional Morphological Phenotyping.

Mannoproteins are non-filamentous glycoproteins localized to the outermost layer of the yeast cell wall. The physiological roles of these structural components have not been completely elucidated due to the limited availability of appropriate tools. As the perturbation of mannoproteins may affect cell morphology, we investigated mannoprotein mutants in Saccharomyces cerevisiae via high-dimensional morphological phenotyping. The mannoprotein mutants were morphologically classified into seven groups using clustering analysis with Gaussian mixture modeling. The pleiotropic phenotypes of cluster I mutant cells (ccw12Δ) indicated that CCW12 plays major roles in cell wall organization. Cluster II (ccw14Δ, flo11Δ, srl1Δ, and tir3Δ) mutants exhibited altered mother cell size and shape. Mutants of cluster III and IV exhibited no or very small morphological defects. Cluster V (dse2Δ, egt2Δ, and sun4Δ) consisted of endoglucanase mutants with cell separation defects due to incomplete septum digestion. The cluster VI mutant cells (ecm33Δ) exhibited perturbation of apical bud growth. Cluster VII mutant cells (sag1Δ) exhibited differences in cell size and actin organization. Biochemical assays further confirmed the observed morphological defects. Further investigations based on various omics data indicated that morphological phenotyping is a complementary tool that can help with gaining a deeper understanding of the functions of mannoproteins.