Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

CONCLUSIONS: The ultrastructural design and biochemical organization of the significantly thickened outer tissues of the gametophytic stem of Hypnodendron menziesii optimizes load bearing of the stem. Hypnodendron menziesii is a bryoid umbrella moss growing in high humid conditions on the forest floors of New Zealand. The erect gametophyte bears up to eight whorls of branches in succession, spreading across the stem that bears the heavy weight of branches with highly hydrated leaves. Our investigation using a combination of light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and TEM-immunolabeling techniques provided novel information on the structural design and biochemical organization of greatly thickened cell walls of epidermal, hypodermal, and outermost cortical tissues, comparing underlying thin-walled cortical tissues in the gametophytic stem. Probing into the ultrastructure of the cell wall architecture of these target tissues by TEM and SEM revealed the cell walls to display a multilamellar organization, in addition to demonstrating the presence of an electron-dense substance in the cell wall, presumably flavonoids. The pattern of distribution and concentration of rhamnogalacturonan, homogalacturonan, and heteromannan, as determined by immunogold labeling, suggests that it is the combination of structural and molecular design of the cell wall that may optimize the mechanical function of the epidermal, hypodermal, and outer cortical tissues. Statistical relationships between the overall thickness of epidermal, hypodermal, and outer cortical cell walls, the lumen area of cells and the percentage area of cell wall occupied in these tissues at different heights of the stem, and thickness of secondary cell wall layers (L1-L4/5) were explored. The results of these analyses unequivocally support the contribution of outer tissues to the mechanical strength of the resilient stem.

Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, \'Ohi\'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan.

Hemicellulose is key in determining the fate of plant cell wall in almost all growth and developmental stages. Nevertheless, there is limited knowledge regarding its involvement in the development and ripening of banana fruit. This study investigated changes in the temporal-spatial distribution of various hemicellulose components, hemicellulose content, activities of the main hydrolysis enzymes, and transcription level of the main hemicellulose-related gene families in banana peels. Both hemicellulose and xylan contents were positively correlated to the fruit firmness observed in our previous study. On the contrary, the xylanase activity was negatively correlated to xylan content and the fruit firmness. The vascular bundle cells, phloem, and cortex of bananas are abundant in xyloglucan, xylan, and mannan contents. Interestingly, the changes in the signal intensity of the CCRC-M104 antibody recognizing non-XXXG type xyloglucan are positively correlated to hemicellulose content. According to RNA-Seq analysis, xyloglucan and xylan-related genes were highly active in the early stages of growth, and the expression of MaMANs and MaXYNs increased as the fruit ripened. The abundance of plant hormonal and growth-responsive cis-acting elements was detected in the 2 kb upstream region of hemicellulose-related gene families. Interaction between hemicellulose and cell wall-specific proteins and MaKCBP1/2, MaCKG1, and MaHKL1 was found. The findings shed light on cell wall hemicellulose\'s role in banana fruit development and ripening, which could improve nutrition, flavor, and reduce postharvest fruit losses.

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of pathogenesis-related (PR) proteins, such as β-1,3-glucanase and peroxidase (POD). This study aims to characterize the physicochemical properties of cell wall-associated POD and β-1,3-glucanase and determine their synergism on the cell wall modification during RWASA2-wheat interaction. The susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5 cultivars were pregerminated and planted under greenhouse conditions, fertilized 14 days after planting, and irrigated every 3 days. The plants were infested with 20 parthenogenetic individuals of the same RWASA2 clone at the 3-leaf stage, and leaves were harvested at 1 to 14 days post-infestation. The Intercellular wash fluid (IWF) was extracted using vacuum filtration and stored at -20 °C. Leaf residues were crushed into powder and used for cell wall components. POD activity and characterization were determined using 5 mM guaiacol substrate and H2O2, monitoring change in absorbance at 470 nm. β-1,3-glucanase activity, pH, and temperature optimum conditions were demonstrated by measuring the total reducing sugars in the hydrolysate with DNS reagent using β-1,3-glucan and β-1,3-1,4-glucan substrates, measuring the absorbance at 540 nm, and using glucose standard curve. The pH optimum was determined between pH 4 to 9, temperature optimum between 25 and 50 °C, and thermal stability between 30 °C and 70 °C. β-1,3-glucanase substrate specificity was determined at 25 °C and 40 °C using curdlan and barley β-1,3-1,4-glucan substrates. Additionally, the β-1,3-glucanase mode of action was determined using laminaribiose to laminaripentaose. The oligosaccharide hydrolysis product patterns were qualitatively analyzed with thin-layer chromatography (TLC) and quantitatively analyzed with HPLC. The method presented in this study demonstrates a robust approach for infesting wheat with RWA, extracting peroxidase and β-1,3-glucanase from the cell wall region and their comprehensive biochemical characterization.

Fertilization relies on pollen mother cells able to transit from mitosis to meiosis to supply gametes. This process involves remarkable changes at the molecular, cellular and physiological levels including (but not limited to) remodelling of the cell wall. During the meiosis onset, cellulose content at the pollen mother cell walls gradually declines with the concurrent deposition of the polysaccharide callose in anther locules. We aim to understand the biological significance of cellulose-to-callose turnover in pollen mother cells walls using electron microscopic analyses of rice flowers. Our observations indicate that in wild type rice anthers, the mitosis-to-meiosis transition coincides with a gradual reduction in the number of cytoplasmic connections called plasmodesmata. A mutant in the Oryza sativa callose synthase GSL5 (Osgsl5-3), impaired in callose accumulation in premeiotic and meiotic anthers, displayed a greater reduction in plasmodesmata frequency among pollen mother cells and tapetal cells suggesting a role for callose in plasmodesmata maintenance. In addition, a significant increase in extracellular distance between pollen mother cells and impaired premeiotic cell shaping was observed in the Osgsl5-3 mutant. The results suggest that callose-to-cellulose turnover during mitosis-meiosis transition is necessary to maintain cell-to-cell connections and optimal extracellular distance among the central anther locular cells. Findings of this study contribute to our understanding of the regulatory influence of callose metabolism during meiosis initiation in flowering plants.

β-1,6-Glucan plays a crucial role in fungal cell walls by linking the outer layer of mannoproteins and the inner layer of β-1,3-glucan, contributing significantly to the maintenance of cell wall rigidity. Therefore, the hydrolysis of β-1,6-glucan by β-1,6-glucanase directly leads to the disintegration of the fungal cell wall. Here, a novel β-1,6-glucanase FlGlu30 was identified from the endophytic Flavobacterium sp. NAU1659 and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of purified FlGlu30 were 50℃ and pH 6.0, resulting in a specific activity of 173.1 U/mg using pustulan as the substrate. The hydrolyzed products of FlGlu30 to pustulan were mainly gentianose within 1 h of reaction. With the extension of reaction time, gentianose was gradually hydrolyzed to glucose, indicating that FlGlu30 is an endo-β-1,6-glucanase. The germination of Magnaporthe oryzae Guy11 spores could not be inhibited by FlGlu30, but the appressorium formation of spores was completely inhibited under the concentration of 250.0 U/mL FlGlu30. The disruptions of cell wall and accumulation of intracellular reactive oxide species (ROS) were observed in FlGlu30-treated M. oryzae Guy11 cells, suggesting the significant importance of β-1,6-glucan as a potential antifungal target and the potential application of FlGlu30. KEY POINTS: • β-1,6-Glucan is a key component maintaining the rigid structure of fungal cell wall. • β-1,6-Glucanase is an antifungal protein with significant potential applications. • FlGlu30 is the first reported β-1, 6-glucanase derived from Flavobacterium.

Tuberculosis (TB) caused by the bacteria Mycobacterium tuberculosis (M. tb), continues to pose a significant worldwide health threat. The advent of drug-resistant strains of the disease highlights the critical need for novel treatments. The unique cell wall of M. tb provides an extra layer of protection for the bacteria and hence only compounds that can penetrate this barrier can reach their targets within the bacterial cell wall. The creation of a reliable machine learning (ML) model to predict the mycobacterial cell wall permeability of small molecules is presented in this work and four ML algorithms, including Random Forest, Support Vector Machines (SVM), k-nearest Neighbour (k-NN) and Logistic Regression were trained on a dataset of 5368 compounds. RDKit and Mordred toolkits were used to calculate features. To determine the most effective model, various performance metrics were used such as accuracy, precision, recall, F1 score, and area under the receiver operating characteristic curve. The best-performing model was further refined with hyperparameter tuning and tenfold cross-validation. The SVM model with filtering outperformed the other machine learning models and demonstrated 80.26% and 81.13% accuracy on the test and validation datasets, respectively. The study also provided insights into the molecular descriptors that play the most important role in predicting the ability of a molecule to pass the M. tb cell wall, which could guide future compound design. The model is available at https://github.com/PGlab-NIPER/MTB_Permeability .

Controlling Campylobacter jejuni during broiler production is a topic of interest from a public health standpoint, as colonized birds can contaminate poultry products during processing and sicken humans if not properly cooked or handled before consumption. The aim of this study was to evaluate dietary yeast cell wall (YCW) as a potential alternative to antibiotic growth promoters with or without a C. jejuni challenge. A total of 2240 day-old Ross 708 males were randomly assigned within 8 experimental groups with a 4 × 2 factorial design, with 4 diets (negative control [CTL-], positive control [CTL+, bacitracin, 50 g/ ton], YCW constant dose [400 g/ton], and YCW step-down dose [SD, 800, 400, and 200 g/ton in the starter, grower, and finisher periods, respectively]) and with or without a Day-16 C. jejuni oral gavage challenge at a 103-colony-forming-units (CFU)/ml dose. Body weights and feed consumption were measured on Days 0, 14, 28, and 41 to determine broiler performance. Ileum tissue samples were collected from 24 birds per treatment on Days 17 and 24 (1 and 8 days postinoculation [PI]) for relative gene expression (RGE) analysis. Cecal content samples were collected from 24 birds per treatment on Days 24, 34, and 42 for C. jejuni enumeration and prevalence calculation. A total of 80 birds per treatment were processed to determine carcass yield on Day 44, and on Day 45, 16 carcass rinsates per treatment were collected for C. jejuni enumeration and prevalence calculation. The interaction between diet and inoculation did not influence growth performance (P > 0.05). However, a diet effect was observed in the starter period where birds fed SD diet had a lower feed conversion ratio than birds fed CTL- diet (P = 0.0165). Additionally, the treatment of birds inoculated with C. jejuni fed with SD had a trend to a lower feed conversion ratio during the grower period (P = 0.0550). The RGE of interleukin 1β and interleukin 10 was similar in all treatments 1 and 8 days PI. The RGE of avian beta defensin 10 was similar in all treatments on Day 1 PI, but different on Day 8 PI (P = 0.0476). All birds inoculated with C. jejuni had similar CFU per milliliter counts in the cecal contents at Days 24, 34, and 42 (P > 0.05), and all birds inoculated with phosphate-buffered saline were negative for C. jejuni after prevalence testing. After processing 1) carcass yield was similar in all treatments (P > 0.05); 2) C. jejuni-inoculated birds fed CTL- had lower CFU per milliliter counts than birds provided CTL+ and constant-dose diets (P = 0.0383); and 3) all birds inoculated with PBS were negative for Campylobacter. Overall, under the conditions of this study, the addition of YCW during a C. jejuni challenge did not have an impact on growth performance, innate immune response, cecal colonization, carcass yield, or carcass colonization after processing.
El papel de la suplementación dietética de paredes celulares de levadura en la respuesta a la inoculación de Campylobacter jejuni en pollos de engorde. El control de Campylobacter jejuni durante la producción de pollos de engorde es un tema de interés desde el punto de vista de la salud pública, ya que las aves colonizadas pueden contaminar los productos avícolas durante el procesamiento y enfermar a los humanos si no se cocinan o manipulan adecuadamente antes del consumo. El objetivo de este estudio fue evaluar la pared celular de levadura dietética (YCW) como una alternativa potencial a los antibióticos promotores del crecimiento con o sin desafío por C. jejuni. Un total de 2240 machos Ross 708 de un día de edad fueron asignados aleatoriamente dentro de ocho grupos experimentales con un diseño factorial 4 × 2, que incluye cuatro dietas (control negativo [CTL–], control positivo [CTL+, bacitracina, 50 g/ton], YCW a dosis constante [400 g/ton] y dosis progresivamente reducida de YCW [SD, 800, 400 y 200 g/ton en los períodos de inicio, crecimiento y finalización, respectivamente]) y con o sin un desafío al día 16 con C. jejuni por sonda oral a una dosis de 103 unidades formadoras de colonias (UFC)/ml. Se midieron los pesos corporales y el consumo de alimento los días 0, 14, 28 y 41 para determinar el rendimiento de los pollos de engorde. Se recolectaron muestras de tejido del íleon de 24 aves por tratamiento en los días 17 y 24 (1 y 8 días después de la inoculación [PI]) para el análisis de la expresión génica relativa (RGE). Se recolectaron muestras de contenido cecal de 24 aves por tratamiento en los días 24, 34 y 42 para la enumeración de C. jejuni y para el cálculo de la prevalencia. Se procesó un total de 80 aves por tratamiento para determinar el rendimiento de la canal en el día 44. En el día 45 se recolectaron 16 enjuagues de la canal por tratamiento para el recuento de C. jejuni y el cálculo de la prevalencia. La interacción entre la dieta y la inoculación no influyó en el rendimiento del crecimiento (P > 0.05). Sin embargo, se observó un efecto de la dieta en el período de iniciación, donde las aves alimentadas con la dieta progresivamente reducida tuvieron una tasa de conversión alimenticia más baja que las aves alimentadas con la dieta control negativa (P = 0.0165). Además, el tratamiento de aves inoculadas con C. jejuni alimentadas con la dieta progresivamente reducida tuvo una tendencia a una tasa de conversión alimenticia más baja durante el período de crecimiento (P = 0.0550). El análisis de expresión génica relativa de la interleucina 1β y la interleucina 10 fue similar en todos los tratamientos a los días uno y ocho después de la inoculación. El análisis de expresión génica relativa de la beta defensina 10 aviar fue similar en todos los tratamientos en el día uno después del desafío, pero diferente en el día ocho después de la inoculación (P = 0.0476). Todas las aves inoculadas con C. jejuni tuvieron recuentos similares de UFC por mililitro en el contenido cecal en los días 24, 34 y 42 (P > 0.05), y todas las aves inoculadas con solución salina amortiguada de fosfatos fueron negativas para C. jejuni después de la prueba de prevalencia. Después del procesamiento 1) el rendimiento de la canal fue similar en todos los tratamientos (P > 0.05); 2) las aves inoculadas con C. jejuni y alimentadas con la dieta control negativa tuvieron recuentos de CFU por mililitro más bajos que las aves que recibieron la dieta control positiva y dietas de dosis constante (P = 0.0383); y 3) todas las aves inoculadas con la solución amortiguada de fosfatos fueron negativas para Campylobacter. En general, bajo las condiciones de este estudio, la adición de la pared celular de levadura dietética durante un desafío con C. jejuni no tuvo un impacto en el rendimiento del crecimiento, en la respuesta inmune innata, en la colonización cecal, en el rendimiento de la canal o la colonización de la canal después del procesamiento.

Caffeine affords several beneficial effects on human health, acting as an antioxidant, anti-inflammatory agent, and analgesic. Caffeine is widely used in cosmetics, but its antimicrobial activity has been scarcely explored, namely against skin infection agents. Dermatophytes are the most common fungal agents of human infection, mainly of skin infections. This work describes the in vitro effect of caffeine during keratinocyte infection by Trichophyton mentagrophytes, one of the most common dermatophytes. The results show that caffeine was endowed with antidermatophytic activity with a MIC, determined following the EUCAST standards, of 8 mM. Caffeine triggered a modification of the levels of two major components of the fungal cell wall, β-(1,3)-glucan and chitin. Caffeine also disturbed the ultrastructure of the fungal cells, particularly the cell wall surface and mitochondria, and autophagic-like structures were observed. During dermatophyte-human keratinocyte interactions, caffeine prevented the loss of viability of keratinocytes and delayed spore germination. Overall, this indicates that caffeine can act as a therapeutic and prophylactic agent for dermatophytosis.