Fungal Cell Wall Research Articles

A novel mannan-specific chimeric antigen receptor M-CAR redirects T cells to interact with Candida spp. hyphae and Rhizopus oryzae spores

ABSTRACT Invasive fungal infections (IFIs) are responsible for elevated rates of morbidity and mortality, causing around of 1.5 million deaths annually worldwide. One of the main causative agents of IFIs is Candida albicans, and non-albicans Candida species have emerged as a spreading global public health concernment. Furthermore, COVID-19 has contributed to a boost in the incidence of IFIs, such as mucormycosis, in which Rhizopus oryzae is the most prevalent causative agent. The effector host immune response against IFIs depends on the activity of T cells, which are susceptible to the regulatory effects triggered by fungal virulence factors. The fungal cell wall plays a crucial role as a virulence factor, and its remodeling compromises the development of a specific T-cell response. The redirection of Jurkat T cells to target Candida spp. by recognizing targets expressed on the fungal cell wall can be facilitated using chimeric antigen receptor (CAR) technology. This study generated an M-CAR that contains an scFv with specificity to α-1,6 mannose backbone of fungal mannan, and the expression of M-CAR on the surface of modified Jurkat cells triggered a strong activation against Candida albicans (hyphae form), Candida tropicalis (hyphae form), Candida parapsilosis (pseudohyphal form), and Candida glabrata (yeast form). Moreover, M-CAR Jurkat cells recognized Rhizopus oryzae spores, which induced high expression of cell activation markers. Thus, a novel Mannan-specific CAR enabled strong signal transduction in modified Jurkat cells in the presence of Candida spp. or R. oryzae.

Serine Hydrolase-Catalyzed Polyol Lipids are Necessary for Rodlet Layer Formation on the Cell Wall of Entomopathogenic Fungi.

Some key secondary metabolism genes are important for driving the infection process of entomopathogenic fungi; however, their chemical substance basis has not been well investigated. Here, mixtures of polyol lipids are discovered, which are synthesized through iterative chain transfer-esterification-hydrolysis cycles catalyzed by serine hydrolase during the release of online highly reducing polyketide intermediates. Importantly, an in vivo gene knockout experiment revealed that the synthesis of polyol lipids is necessary for rodlet layer formation on the cell wall of Beauveria bassiana. Our work uncovers an unexpected way for the synthesis of polyol lipids and illuminates a new perspective on their part in significant physiological processes in entomopathogenic fungi.

Silver Nanoparticles Produced Extracellularly by Sclerotinia sclerotiorum

Abstract Background: Sclerotinia sclerotiorum is one of the most important fungal species capable of manufacturing silver nanoparticles (AgNPs), which are formed through the biosynthesis process extracellular from silver nitrate solution (AgNo3) to the culture medium using a fungal cell filter; this study aims to explore the biosynthesis of AgNPs using S. sclerotiorum. Materials and Methods: AgNPs were produced by adding AgNo3 to the culture and then incubating for 72 h at 37°C. The nanoparticles were formed inside the cytoplasm and on the outer surface of the S. sclerotiorum fungus. These molecules were characterized using ultraviolet radiation, and the highest absorbance of AgNPs for S. sclerotiorum fungus was measured at 450 nm. AgNPs were also characterized using infrared analysis (Fourier transform infrared) and scanning electron microscope (SEM) to determine the size and crystal structure of these molecules. Results: S. sclerotiorum is a well-known phytopathogenic fungus with significant potential beyond its role as a plant pathogen. Various isolates of this fungus have diverse applications in agriculture, biotechnology, and nanotechnology. Notably, these isolates have demonstrated potential in reducing silver ions (Ag+) to (AgNPs), highlighting their promise in nanoparticle synthesis. In addition, the intensity of the absorption peak associated with the nanoparticles was found to increase over time, further supporting their potential for research and application. Conclusion: AgNPs produced by S. sclerotiorum is predominantly spherical, ranging from 5 to 50 nm, and are stabilized by a capping agent. These nanoparticles, found mainly on the fungal cell walls, likely result from fungal enzyme activity. Their antifungal properties are due to their interference with microbial DNA and disruption of metabolic processes, highlighting their potential as effective antifungal agents.

Massive gene loss in the fungus Sporothrix epigloea accompanied a shift to life in a glucuronoxylomannan-based gel matrix.

Fungi are well known for their ability to both produce and catabolize complex carbohydrates to acquire carbon, often in the most extreme of environments. Glucuronoxylomannan (GXM)-based gel matrices are widely produced by fungi in nature and though they are of key interest in medicine and pharmaceuticals, their biodegradation is poorly understood. Though some organisms, including other fungi, are adapted to life in and on GXM-like matrices in nature, they are almost entirely unstudied, and it is unknown if they are involved in matrix degradation. Sporothrix epigloea is an ascomycete fungus that completes its life cycle entirely in the short-lived secreted polysaccharide matrix of a white jelly fungus, Tremella fuciformis. To gain insight into how S. epigloea adapted to life in this unusual microhabitat, we compared the predicted protein composition of S. epigloea to that of 21 other Sporothrix species. We found that the genome of S. epigloea is smaller than that of any other sampled Sporothrix, with widespread functional gene loss, including those coding for serine proteases and biotin synthesis. In addition, many predicted CAZymes degrading both plant and fungal cell wall components were lost while a lytic polysaccharide monooxygenase (LPMO) with no previously established activity or substrate specificity, appears to have been gained. Phenotype assays suggest narrow use of mannans and other oligosaccharides as carbon sources. Taken together, the results suggest a streamlined machinery, including potential carbon sourcing from GXM building blocks, facilitates the hyperspecialized ecology of S. epigloea in the GXM-like milieu.

Unveiling the potential of Aspergillus terreus SJP02 for zinc remediation and its driving mechanism

In present study, 15 morphologically different fungi isolated from rhizopheric soils of an industrial area were screened for their Zn2+ removal efficiency from aqueous solution. Isolate depicting highest potential was molecularly identified as Aspergillus terreus SJP02. Effect of various process parameters viz. biosorbent dose, contact time, temperature, agitation rate, pH and initial Zn2+ concentration on the fungal sorption capacity were studied. The biosorbent exhibited maximum Zn2+ sorption capacity of 10.7 ± 0.2 mg g− 1 in 60 min. Desorption studies showed 71.46% Zn2+ recovery rate in 120 min with 0.01 N HNO3, indicating efficient metal recovery for reuse and subsequent reutilization of spent mycosorbents. Acid digestion study suggested adsorption being the primary mechanism accounting for 87% Zn2+removal. It was further confirmed by the FE-SEM and EDX analysis. FTIR analysis suggested involvement of amino, hydroxyl, carbonyl, and phosphate functional groups of fungal cell wall in adsorption. The experimental results were in accordance with the tested isotherm and kinetic models, and suggested the role of physical adsorption for Zn2+ removal. Noteworthy, the present study showed better sorption capacity in considerably shorter equilibration time compared to previous reports and advocate potential utilization of A. terreus SJP02 for bioremediation of Zn2+ contaminated wastewater at industrial scale.

CSE-8, a filamentous fungus-specific Shr3-like chaperone, facilitates endoplasmic reticulum exit of chitin synthase CHS-3 (class I) in Neurospora crassa

Chitin is a crucial structural polysaccharide in fungal cell walls, essential for maintaining cellular plasticity and integrity. Its synthesis is orchestrated by chitin synthases (CHS), a major family of transmembrane proteins. In Saccharomyces cerevisiae, the cargo receptor Chs7, belonging to the Shr3-like chaperone family, plays a pivotal role in the exit of Chs3 from the endoplasmic reticulum (ER) and its subsequent activity in the plasma membrane (PM). However, the auxiliary machinery responsible for CHS trafficking in filamentous fungi remains poorly understood. The Neurospora crassa genome encodes two orthologues of Chs7: chitin synthase export (CSE) proteins CSE-7 (NCU05720) and CSE-8 (NCU01814), both of which are highly conserved among filamentous fungi. In contrast, yeast forms only possess a single copy CHS export receptor. Previous research highlighted the crucial role of CSE-7 in the localization of CHS-4 at sites of cell wall synthesis, including the Spitzenkörper (SPK) and septa. In this study, CSE-8 was identified as an export protein for CHS-3 (class I). In the Δcse-8 knockout strain of N. crassa, CHS-3-GFP fluorescence was absent from the SPK or septa, indicating that CSE-8 is required for the exit of CHS-3 from the ER. Additionally, sexual development was disrupted in the Δcse-8 strain, with 20% of perithecia from homozygous crosses exhibiting two ostioles. A Δcse-7;Δcse-8 double mutant strain showed reduced N-acetylglucosamine (GlcNAc) content and decreased radial growth. Furthermore, the loss of cell polarity and the changes in subcellular distribution of CSE-8-GFP and CHS-3-GFP observed in hyphae under ER stress induced by the addition of tunicamycin and dithiothreitol reinforce the hypothesis that CSE-8 functions as an ER protein. The current evidence suggests that the biogenesis of CHS exclusive to filamentous fungi may involve pathways independent of CSE-mediated receptors.

Reevaluating the Value of (1,3)-β-D-Glucan for the Diagnosis of Intra-Abdominal Candidiasis in Critically Ill Patients: Current Evidence and Future Directions

Intra-abdominal candidiasis (IAC) is associated with significant diagnostic and therapeutic challenges in critically ill patients. Traditional fungal cultures are slow, delaying appropriate antifungal treatment. (1,3)-β-D-glucan (BDG), a component of the fungal cell wall, has emerged as a potential biomarker for IAC, but its use in ICU settings is complicated by frequent false-positives results from invasive procedures and underlying conditions. This review examines the diagnostic value of BDG when present in serum and peritoneal fluid. While serum BDG is effective for excluding invasive fungal infections like candidemia, its specificity for IAC remains low in critically ill patients. Recent studies suggest that BDG levels in peritoneal fluid may provide better diagnostic accuracy, distinguishing IAC from bacterial peritonitis with higher specificity. We discuss the advantages, limitations, and practical aspects of BDG testing, emphasizing the potential of peritoneal BDG as a complementary tool. Further research is needed to refine diagnostic thresholds, validate its clinical utility, and establish the role of peritoneal BDG in improving timely, targeted antifungal treatment for IAC.

Synthesis and evaluation of the antifungal and antibiofilm potential of aminochalcones.

Candida is a commensal fungus of clinical interest that commonly lives in oral cavity and intestine but can become an opportunist microrganism and cause severe infections. A serie of 10 aminochalcones were designed and synthetized to obtain compounds anti-Candida with potent and broad-spectrum activity. The most active compound J34 demonstrated excellent in vitro activity against Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata and Candida krusei with minimum inhibitory concentration between 1.9 and 7.8µg/mL. The association of aminochalcone J34 with amphotericin B demonstrated synergistic effect against C. albicans, with Fractional Inhibiroty Concentration Index (FICI) value of 0.5. Subinhibitory concentration of J34 inhibited the C. albicans adhesion to human keratinocytes. Treatment with J34 reduced C. albicans biofilm formation, as well as acts on preformed biofilm in concentration-dependent mode. Time-kill curve demonstrated that J34 had fungicidal action after 12h of treatment. Preliminary mechanism of action study showed J34 interacts with membrane ergosterol but does not act on fungal cell wall of C. albicans. In additon, in vivo studies using Galleria mellonella indicated low toxic effect of chalcone J34 after 72h of treatment.

Botrytis cinerea PMT4 Is Involved in O-Glycosylation, Cell Wall Organization, Membrane Integrity, and Virulence.

Proteins found within the fungal cell wall usually contain both N- and O-oligosaccharides. N-glycosylation is the process where these oligosaccharides (hereinafter: glycans) are attached to asparagine residues, while in O-glycosylation the glycans are covalently bound to serine or threonine residues. The PMT family is grouped into PMT1, PMT2, and PMT4 subfamilies. Using bioinformatics analysis within the Botrytis cinerea genome database, an ortholog to Saccharomyces cerevisiae Pmt4 and other fungal species was identified. The aim of this study was to assess the relevance of the bcpmt4 gene in B. cinerea glycosylation. For this purpose, the bcpmt4 gene was disrupted by homologous recombination in the B05.10 strain using a hygromycin B resistance cassette. Expression of bcpmt4 in S. cerevisiae ΔScpmt4 or ΔScpmt3 null mutants restored glycan levels like those observed in the parental strain. The phenotypic analysis showed that Δbcpmt4 null mutants exhibited significant changes in hyphal cell wall composition, including reduced mannan levels and increased amounts of chitin and glucan. Furthermore, the loss of bcpmt4 led to decreased glycosylation of glycoproteins in the B. cinerea cell wall. The null mutant lacking PMT4 was hypersensitive to a range of cell wall perturbing agents, antifungal drugs, and high hydrostatic pressure. Thus, in addition to their role in glycosylation, the PMT4 is required to virulence, biofilm formation, and membrane integrity. This study adds to our knowledge of the role of the B. cinerea bcpmt4 gene, which is involved in glycosylation and cell biology, cell wall formation, and antifungal response.

A microbial natural product fractionation library screen with HRMS/MS dereplication identifies new lipopeptaibiotics against Candida auris.

The rise of drug-resistant fungal pathogens, including Candida auris , highlights the urgent need for novel antifungal therapies. We developed a cost-effective platform combining microbial extract prefractionation with rapid MS/MS-bioinformatics-based dereplication to efficiently prioritize new antifungal scaffolds. Screening C. auris and C. albicans revealed novel lipopeptaibiotics, coniotins, from Coniochaeta hoffmannii WAC11161, which were undetectable in crude extracts. Coniotins exhibited potent activity against critical fungal pathogens on the WHO Fungal Priority Pathogens List, including C. albicans , C. neoformans , multidrug-resistant C. auris , and Aspergillus fumigatus , with high selectivity and low resistance potential. Coniotin A targets β-glucan, compromising fungal cell wall integrity, remodelling, and sensitizing C. auris to caspofungin. Identification of a PKS-NRPS biosynthetic gene cluster further enables the discovery of related clusters encoding potential novel lipopeptaibiotics. This study demonstrates the power of natural product prefractionation in uncovering bioactive scaffolds and introduces coniotins as promising candidates for combating multidrug-resistant fungal pathogens.

Fungal effector genes involved in the suppression of chitin signaling as novel targets for the control of powdery mildew disease via a nontransgenic RNA interference approach.

Chitin is a crucial component of fungal cell walls and an effective elicitor of plant immunity; however, phytopathogenic fungi have developed virulence mechanisms to counteract the activation of this plant defensive response. In this study, the molecular mechanism of chitin-induced suppression through effectors involved in chitin deacetylases (CDAs) and their degradation (EWCAs) was investigated with the idea of developing novel dsRNA-biofungicides to control the cucurbit powdery mildew caused by Podosphaera xanthii. The molecular mechanisms associated with the silencing effect of the PxCDA and PxEWCAs genes were first studied through dsRNA cotyledon infiltration assays, which revealed a ≈80% reduction in fungal biomass and a 50% decrease in gene expression. To assess the impact on powdery mildew disease control, in vitro and in planta assays were carried out in growth chamber and glasshouse experiments, with ≈50% reduction in disease symptoms after 8 days postinoculation (dpi) in leaf discs and 12 dpi in plants' leaves, respectively. This control was extended for 21 days when the dsRNAs were protected on the carbon dot nanocarriers. Additionally, the uptake of the dsRNAs by fungal spores was observed 12 h postapplication via confocal microscopy, and efficient processing of dsRNAs into siRNAs by the melon RNAi machinery was observed 24 h postspraying through sRNA-seq. This study highlights notable advancements in environmentally friendly disease management, and features the technological potential of RNA-based fungicides together with nanotechnology for cucurbit powdery mildew control. © 2025 Society of Chemical Industry.

Plant-based Therapeutics for Aspergillus Co-infections in COVID-19 Patients Illuminating Phytochemical Mechanisms

The emergence of COVID-19 has highlightedthe issue of secondary fungal infections, such as COVID-19-Associated invasive Pulmonary Aspergillosis (CAPA), in critically ill patients. Continuous use of therapies employing steroids and immune-modulating agents has increased the risk of co-infections resulting from fungi, further weakening the respiratory systems of patients and leading to complex situations such as multi-organ failure and elevated mortality. The increasing occurrence of antifungal resistance in Aspergillus strains has made the management of these infections challenging, highlighting the need for novel and effective antifungal agents. Plants and their bioactive compounds offer promising alternatives to conventional antifungal medications. Phytochemicals such as 1,8-cineole from Eucalyptus, thymol from commercial extracts, and cinnamaldehyde from cinnamon have demonstrated significant antifungal activities by targeting fungal cell walls, membranes, and gene expression. Additionally, compounds like eugenol from cloves and citral from lemongrass exhibit potent antimicrobial effects by disrupting cellular integrity. However, challenges such as variability in phytochemical content, limited clinical data, and potential drug interactions must be addressed. This review explores the potential of plant-based medicines for treating aspergillosis, emphasizing the need for further research to validate their effectiveness, safety, and optimal dosages through clinical studies.

Identification, Biocontrol and Plant Growth Promotion Potential of Endophytic Streptomyces sp. a13.

Medicinal plants often harbour various endophytic actinomycetia, which are well known for their potent antimicrobial properties and plant growth-promoting traits. In this study, we isolated an endophytic actinomycetia, A13, from the leaves of tea clone P312 from the MEG Tea Estate, Meghalaya, India. The isolate A13 was identified as Streptomyces sp. A13 through whole genome sequencing (WGS) and 16S rRNA sequencing, showing 88% (ANI; Average Nucleotide Identity) and 99.78% sequence similarity with Streptomyces olivaceus. The strain A13 exhibited a prominent broad-spectrum antifungal activity against nine phytopathogens. It was observed that the ethyl acetate (EtAc) extract of A13 inhibits the spore germination rate of phytopathogen Nigrospora sphaerica (NSP) and also damages the fungal cell wall and cell structure. Additionally, the A13 strain exhibits several plant growth-promoting (PGP) traits, such as nitrogen fixation, ammonia production (4.7µmol/ml), indole-acetic acid (IAA) production (8.91µg/ml), siderophore production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity Gas chromatography mass spectrometry (GC-MS) analysis revealed that Phenol, 3,5-bis(1,1-dimethylethyl) was found to be the major chemical constituent in the EtAc extract of the A13 strain, accounting for 50.15% of the area percentage. Whole genome sequencing and subsequent genome analysis utilizing bioinformatics techniques such as Antibiotics & Secondary Metabolite Analysis SHell (antiSMASH) and Rapid Annotation using Subsystem Technology (RAST) revealed a wide array of biologically active secondary metabolite biosynthesis gene clusters (smBGCs) with different physiologically significant roles. These findings emphasize the potential of the A13 strain as a biocontrol agent with the capability to enhance plant growth and prevent diseases.

The Significance of Mono- and Dual-Effective Agents in the Development of New Antifungal Strategies.

Invasive fungal infections (IFIs) pose significant challenges in clinical settings, particularly due to their high morbidity and mortality rates. The rising incidence of these infections, coupled with increasing antifungal resistance, underscores the urgent need for novel therapeutic strategies. Current antifungal drugs target the fungal cell membrane, cell wall, or intracellular components, but resistance mechanisms such as altered drug-target interactions, enhanced efflux, and adaptive cellular responses have diminished their efficacy. Recent research has highlighted the potential of dual inhibitors that simultaneously target multiple pathways or enzymes involved in fungal growth and survival. Combining pharmacophores, such as lanosterol 14α-demethylase (CYP51), heat shock protein 90 (HSP90), histone deacetylase (HDAC), and squalene epoxidase (SE) inhibitors, has led to the development of compounds with enhanced antifungal activity and reduced resistance. This dual-target approach, along with novel chemical scaffolds, not only represents a promising strategy for combating antifungal resistance but is also being utilized in the development of anticancer agents. This review explores the development of new antifungal agents that employ mono-, dual-, or multi-target strategies to combat IFIs. We discuss emerging antifungal targets, resistance mechanisms, and innovative therapeutic approaches that offer hope in managing these challenging infections.

Enhanced antifungal activity of siRNA-loaded anionic liposomes against the human pathogenic fungus Aspergillus fumigatus.

We developed siRNA-loaded anionic liposomes, co-encapsulating low-dose amphotericin B, to enhance siRNA penetration through the fungal cell wall of Aspergillus fumigatus. Targeting mRNAs of three key genes, these liposomes visibly inhibited fungal growth, demonstrating for the first time the antifungal potential of siRNA against human fungal pathogens.

Antifungal Potency of Biosynthesized Silver Nanoparticles Derived from Marine Diatoms Against Multidrug‐Resistant Candida auris and Pichia kudriavzevii

AbstractCandida auris and Pichia kudriavzevii are emerging multidrug‐resistant fungal pathogens that pose a significant threat to public health. The limited efficacy of conventional antifungals against these species has prompted the development of novel antifungal compounds. In recent years, silver nanoparticles (AgNPs) synthesized using marine diatoms have held promise as potent antifungal agents. In this study, three marine diatom species (Chaetoceros spp., Skeletonema spp., and Thalassiosira spp.) were utilized for the biosynthesis of AgNPs (Ag‐DE/NPs). The biosynthesis was confirmed by a color change of the culture from colorless to brown and further validated by UV–vis spectroscopy, showing distinct surface plasmon resonance peaks at 425, 430, and 440 nm, respectively. Comprehensive characterization using FTIR, XRD, DLS, and SEM revealed the functionalized nature, crystalline structure, particle size, and surface morphology of the Ag‐DE/NPs. The antifungal efficacy of these AgNPs was evaluated against 20 clinical isolates and 2 reference strains of C. auris and P. kudriavzevii, which exhibited high resistance to fluconazole. AgNPs synthesized from Chaetoceros spp. displayed the lowest geometric mean minimum inhibitory concentrations (0.23 µg/mL for C. auris and 0.19 µg/mL for P. kudriavzevii), showing a >250‐fold greater potency compared to fluconazole and comparable efficacy to amphotericin B. Growth curve analysis and sorbitol supplementation assays indicated that Ag‐DE/NPs disrupt fungal cell walls, while SEM imaging and ergosterol quantitation confirmed membrane damage and sterol depletion. These findings underscore the potential of Ag‐DE/NPs, particularly those synthesized from Chaetoceros spp., as promising candidates for combating drug‐resistant fungal infections.

A conserved fungal Knr4/Smi1 protein is crucial for maintaining cell wall stress tolerance and host plant pathogenesis.

Filamentous plant pathogenic fungi pose significant threats to global food security, particularly through diseases like Fusarium Head Blight (FHB) and Septoria Tritici Blotch (STB) which affects cereals. With mounting challenges in fungal control and increasing restrictions on fungicide use due to environmental concerns, there is an urgent need for innovative control strategies. Here, we present a comprehensive analysis of the stage-specific infection process of Fusarium graminearum in wheat spikes by generating a dual weighted gene co-expression network (WGCN). Notably, the network contained a mycotoxin-enriched fungal module (F12) that exhibited a significant correlation with a detoxification gene-enriched wheat module (W12). This correlation in gene expression was validated through quantitative PCR. By examining a fungal module with genes highly expressed during early symptomless infection that was correlated to a wheat module enriched in oxidative stress genes, we identified a gene encoding FgKnr4, a protein containing a Knr4/Smi1 disordered domain. Through comprehensive analysis, we confirmed the pivotal role of FgKnr4 in various biological processes, including oxidative stress tolerance, cell cycle stress tolerance, morphogenesis, growth, and pathogenicity. Further studies confirmed the observed phenotypes are partially due to the involvement of FgKnr4 in regulating the fungal cell wall integrity pathway by modulating the phosphorylation of the MAP-kinase MGV1. Orthologues of the FgKnr4 gene are widespread across the fungal kingdom but are absent in other Eukaryotes, suggesting the protein has potential as a promising intervention target. Encouragingly, the restricted growth and highly reduced virulence phenotypes observed for ΔFgknr4 were replicated upon deletion of the orthologous gene in the wheat fungal pathogen Zymoseptoria tritici. Overall, this study demonstrates the utility of an integrated network-level analytical approach to pinpoint genes of high interest to pathogenesis and disease control.

Expression and functional analysis of mouse chitinases without the ZZ domain of Staphylococcus aureus Protein A.

Chitinase plays a role in mammalian immune responses, particularly in the degradation of fungal cell walls. The aim of the present study was to express and characterize recombinant mouse chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) without the ZZ domain, a domain that may interfere with immunological analyses. We successfully expressed recombinant chitinases without the ZZ domain (Chit1-V5-His and AMCase-V5-His) as a soluble protein from an expression vector pET21a in the Escherichia coli Rosetta-gami B (DE3) strain. Chit1-V5-His exhibited chitinolytic activity similar to that of ProteinA-Chit1-V5-His (a recombinant Chit1 with the ZZ domain) and natural Chit1, both with synthetic and natural substrates. Differential scanning fluorimetry and thermal stability assays revealed that Chit1-V5-His retained functional stability comparable to that of ProteinA-Chit1-V5-His, although ProteinA-Chit1-V5-His was more thermally stable. AMCase-V5-His demonstrated prominent chitinolytic activity at pH 2.0, aligning with the properties of natural AMCase. Owing to the lack of the ZZ domain that potentially binds to immunoglobulin G Fc region, Chit1-V5-His and AMCase-V5-His are advantageous tools for immunological analyses, as they do not block the Fc receptor-mediated phagocytosis of fungi by polymorphonuclear neutrophils and macrophages. Thus, this expression system effectively produces functional chitinases, facilitating further studies on their roles in mammalian immunity.

ГИДРОЛИЗУЮЩАЯ АКТИВНОСТЬ ЦГТаз БАКТЕРИЙ РОДА PAENIBACILLUS ПО ОТНОШЕНИЮ К α- И β-ГЛИКАНАМ

The cell wall of micromycetes is crucial for the shape and function of cells, as well as for their protection from environmental influences and contains up to 80-90 % of polysaccharides with different types of bonds. Cyclodextrin glucanotransferase microorganisms were tested and the following strains were selected Paenibacillus ehimensis IB-739, Paenibacillus illinoisensis IB-1087, Paenibacillus sp. IB-663, Paenibacillus macerans IB-I4. These strains were able to hydrolyze homopolysaccharides (starch, carboxyme-thylcellulose) and heteropolysaccharides (glucomannan, galactomannan). Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is a multifunctional enzyme capable of catalyzing various types of reactions: cyclization, binding, disproportionation and hydrolysis. It was found that strains and their preparations of CGTase, in addition to cyclizing activity, have a hydrolyzing ability against α- and β-glicans. The method of cluster analysis for strains revealed substrate preferences during hydrolysis in favor of α glicans (P. ehimensis IB-739, Paenibacillus sp. IB-663) or β-glicans (P. illinoisensis IB-1087, P. macerans IB-I4). The advantage of the preparative form of the CGTase enzyme when exposed to the components of the fungal cell wall (α-glicans and β-glicans) in comparison with the intact effect of cellular hydrolases is shown. The culture of P. macerans IB-I4, whose CGTase equally hydrolyzes all the presented polymers, turned out to be the most active in terms of the degree of destruction of glicans. The prospect of using CGTase enzyme preparations as agents in the fight against phytopathogenic fungi is discussed.

Mixed DAMP/MAMP oligosaccharides promote both growth and defense against fungal pathogens of cucumber.

Plants recognize a variety of environmental molecules, thereby triggering appropriate responses to biotic or abiotic stresses. Substances containing microbes-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) are representative inducers of pathogen resistance and damage repair, thus treatment of healthy plants with such substances can pre-activate plant immunity and cell repair functions. In this study, the effects of DAMP/MAMP oligosaccharides mixture (Oligo-Mix) derived from plant cell wall (cello-oligosaccharide and xylo-oligosaccharide), and fungal cell wall (chitin-oligosaccharide) were examined in cucumber. Treatment of cucumber with Oligo-Mix promoted root germination and plant growth, along with increased chlorophyll contents in the leaves. Oligo-Mix treatment also induced typical defense responses such as MAP kinase activation and callose deposition in leaves. Pretreatment of Oligo-Mix enhanced disease resistance of cucumber leaves against pathogenic fungi Podosphaera xanthii (powdery mildew) and Colletotrichum orbiculare (anthracnose). Oligo-Mix treatment increased the induction of hypersensitive cell death around the infection site of pathogens, which inhibited further infection and the conidial formation of pathogens on the cucumber leaves. RNA-seq analysis revealed that Oligo-Mix treatment upregulated genes associated with plant structural reinforcement, responses to abiotic stresses and plant defense. These results suggested that Oligo-Mix has beneficial effects on growth and disease resistance in cucumber, making it a promising biostimulant for agricultural application.