Expression of a novel NaD1 recombinant antimicrobial peptide enhances antifungal and insecticidal activities.

This study aimed to increase the antifungal and insecticidal activities of NaD1, as an antimicrobial peptides (AMP), by improving its interaction with the fungal cell wall and chitin monomeric units in insect midguts. Hence, the chitin-binding domains (CBDs) of wheat germ agglutinin protein (WGA) were fused to either N- or C-terminus of NaD1 generating transgenic Nicotiana tabacum hairy roots (HRs). Molecular assessments confirmed the integration of NaD1 transgenes, their transcription and production of recombinant peptides in the HR lines. Total protein of (CBD)4-NaD1 and NaD1-(CBD)4 transgenic lines inhibited the growth of Pyricularia oryzae mycelium, suggesting that fusion of CBD to NaD1 can increase NaD1 half-life, leading to higher affinity toward cell wall chitin. Furthermore, feeding the third-instar larvae of Chilo suppressalis with both (CBD)4-NaD1 and NaD1-(CBD)4 extracts exhibited a higher mortality rate. Both NaD1-CBDs caused a significant decrease in trypsin (TRY) and chymotrypsin (CTR) activities in the larvae, while enhancing the activity of antioxidant enzymes CAT, POD, APX, and SOD. Therefore, feeding the larvae by total extract of NaD1-(CBD)4 and (CBD)4-NaD1 HR lines probably increased affinity to midgut chitin in C. suppressalis, enhancing insecticidal activities. Overall, the results indicate that recombinant peptides are effective in enhancing fungal and insect resistance.

Chitinase and Deacetylase-Based Chitin-Degrading Bacteria: One-Pot Cascade Bioconversion of Chitin to Chitooligosaccharides.

Cascade conversion of chitin into soluble and functional chitooligosaccharides has gained great attention. However, the biotransformation route is still limited to the low catalytic performances of chitin deacetylases (CDAs) and complicated procedures. In this study, a CDA from Arthrobacter sp. Jub115 (ArCDA) was identified and characterized, which showed a higher catalytic stability than the reported CDAs, with residual activity of 80.49%, 71.12%, and 56.09% after incubation at 30, 35, and 40 °C for 24 h, respectively. Additionally, ArCDA was identified to have a broad substrate spectrum toward ß-chitin and N-acetyl chitooligosaccharides. Moreover, an engineered chitin-degrading bacteria (CDB) with cell-surface-displayed deacetylase ArCDA and chitinase SaChiB was constructed to simplify catalysis procedures, facilitating the chitobiose production of 294.30 ± 16.43 mg/L in 10 h. This study not only identified a CDA with the desirable catalytic performance but also provided a strategy for constructing CDB, facilitating the high-value utilization of chitin.

Engineering Bacterial Chitinases for Industrial Application: From Protein Engineering to Bacterial Strains Mutation! A Comprehensive Review of Physical, Molecular, and Computational Approaches.

Bacterial chitinases are integral in breaking down chitin, the natural polymer in crustacean and insect exoskeletons. Their increasing utilization across various sectors such as agriculture, waste management, biotechnology, food processing, and pharmaceutical industries highlights their significance as biocatalysts. The current review investigates various scientific strategies to maximize the efficiency and production of bacterial chitinases for industrial use. Our goal is to optimize the heterologous production process using physical, molecular, and computational tools. Physical methods focus on isolating, purifying, and characterizing chitinases from various sources to ensure optimal conditions for maximum enzyme activity. Molecular techniques involve gene cloning, site-directed mutation, and CRISPR-Cas9 gene editing as an approach for creating chitinases with improved catalytic activity, substrate specificity, and stability. Computational approaches use molecular modeling, docking, and simulation techniques to accurately predict enzyme-substrate interactions and enhance chitinase variants' design. Integrating multidisciplinary strategies enables the development of highly efficient chitinases tailored for specific industrial applications. This review summarizes current knowledge and advances in chitinase engineering to serve as an indispensable guideline for researchers and industrialists seeking to optimize chitinase production for various uses.

Fgk3, a Glycogen Synthase Kinase, Regulates Chitin Synthesis through the Carbon Catabolite Repressor FgCreA in Fusarium graminearum.

The glycogen synthase kinase-3 (GSK3) orthologs are well-conserved in eukaryotic organisms. However, their functions remain poorly characterized in filamentous fungi. In our previous study, we unveiled the function of Fgk3, the GSK3 ortholog, in glycogen metabolism in Fusarium graminearum, the causal agent of Fusarium head blight. Interestingly, the fgk3 mutant was unstable and tended to produce fast-growing suppressors, including secondary suppressors. Using whole-genome sequencing, we identified suppressor mutations in FgCHS5, FgFKS1, FgCREA, FgSSN6, FgRGR1, and FgPP2A in nine primary and four secondary suppressors. Subsequently, we validated that deletion of FgCHS5 or FgCREAΔH253 mutation partially suppressed the defects of fgk3 in vegetative growth and cell wall integrity, suggesting that Fgk3 may regulate the chitin synthesis through FgCreA-mediated transcriptional regulation in F. graminearum. Accordingly, the FGK3 deletion led to hyphal swelling with abnormal chitin deposition, and deletion of FGK3 or FgCREA caused the upregulation of the expression of chitin synthases FgCHS5 and FgCHS6. The interaction between Fgk3 and FgCreA was verified by Yeast two-hybrid and Co-Immunoprecipitation assays. More importantly, we verified that the nuclear localization and protein stability of FgCreA relies on the Fgk3 kinase, while the H253 deletion facilitated the re-localization of FgCreA to the nucleus in the fgk3 mutant background, potentially contributing to the suppression of the fgk3 mutant's defects. Intriguingly, the ΔH253 mutation of FgCreA, identified in suppressor mutant S3, is adjacent to a conserved phosphorylation site, S254, suggesting that this mutation may inhibit the S254 phosphorylation and promote the nuclear localization of FgCreA. Collectively, our findings indicate that the glycogen synthase kinase Fgk3 regulates the chitin synthesis through the carbon catabolite repressor FgCreA in F. graminearum.

Knockdown of the glucosamine-6-phosphate N-acetyltransferase gene by RNA interference enhances the virulence of entomopathogenic fungi against rice leaffolder Cnaphalocrocis medinalis.

Insect cuticle acts as a first line of defense and a physical protective barrier against entomopathogens. Chitin biosynthesis pathway plays a crucial role in chitin formation in the cuticle of insects. Glucosamine-6-phosphate N-acetyltransferase (GNA) is a key enzyme in insect chitin biosynthesis that regulate the chitin formation. However, how GNA-mediated cuticle metabolism influences virulence of entomopathogenic fungi is still unknown. In this study, CmGNA gene was cloned and characterized from the rice leaffolder Cnaphalocrocis medinalis. The CmGNA contains an open read frame (ORF) 600 nucleotides, encoding 199 amino acids with an isoelectric point of 8.65 and a molecular weight of 22.30 kDa. The expression profile showed that CmGNA was highly expressed in 4th instar larvae and in the cuticle. Here, we also reported the impact of CmGNA gene and entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, on expression pattern of chitin biosynthesis genes, feeding behavior, survival rate and average body weight of infected larvae, phenotypic deformities, rate of pupation, and adult emergence. Our results showed that knockdown of CmGNA and application of M. anisopliae and B. bassiana three days after RNA interference (RNAi) significantly decreased the expression of CmGNA and other associated genes, reduced feeding efficiency and survival rate, and caused loss of average body weight, less rate of pupation and adult emergence of infected larvae. Knockdown of CmGNA gene also increased the lethality of larvae caused by M. anisopliae and B. bassiana and resulted in significantly phenotypic deformities of infected larvae. Our findings illustrated that RNAi-mediated CmGNA knockdown disturbed the chitin synthesis genes that led to enhancing the virulence of M. anisopliae and B. bassiana, which can provide us new insights to develop novel biocontrol strategies against C. medinalis.

Affinity molecular assay for detecting Candida albicans using chitin affinity and RPA-CRISPR/Cas12a.

Invasive fungal infections (IFIs) pose a significant threat to immunocompromised individuals, leading to considerable morbidity and mortality. Prompt and accurate diagnosis is essential for effective treatment. Here we develop a rapid molecular diagnostic method that involves three steps: fungal enrichment using affinity-magnetic separation (AMS), genomic DNA extraction with silicon hydroxyl magnetic beads, and detection through a one-pot system. This method, optimized to detect 30 CFU/mL of C. albicans in blood and bronchoalveolar lavage (BAL) samples within 2.5 h, is approximately 100 times more sensitive than microscopy-based staining. Initial validation using clinical samples showed 93.93% sensitivity, 100% specificity, and high predictive values, while simulated tests demonstrated 95% sensitivity and 100% specificity. This cost-effective, highly sensitive technique offers potential for use in resource-limited clinical settings and can be easily adapted to differentiate between fungal species and detect drug resistance.

Hyphal swelling induced in the phagosome of macrophages.

Macrophages play critical protective roles as sentinels of the innate immune system against fungal infection. It is therefore important to understand the dynamics of the interaction between these phagocytes and their fungal prey. We show here that many of the hyphal apices formed by Candida albicans within the macrophage ceased elongating, and apical and sub-apical hyphal compartments became swollen. Swollen hyphal cell compartments assimilated less Lysotracker-Red than non-swollen compartments, suggesting they had enhanced viability. Staining with florescent dyes suggested that there were higher levels of ß-glucan and chitin in internalized fungal filaments compared to non-internalized hyphae, suggesting active cell wall remodelling within macrophages. These observations suggest that the stresses imposed by macrophages upon the fungus lead to changes in cell wall composition, inhibition of polarised growth and the induction of swelling in hyphal compartments, and that this can prevent or delay loss of viability of hyphal cells within the phagocyte.

Multi-omics analysis reveal the fall armyworm Spodoptera frugiperda tolerate high temperature by mediating chitin-related genes.

Climate change facilitates the rapid invasion of agricultural pests, threatening global food security. The fall armyworm Spodoptera frugiperda is a highly polyphagous migratory pest tolerant to high temperatures, allowing its proliferation in harsh thermal environments. We aimed to demonstrate mechanisms of its high-temperature tolerance, particularly transcriptional and metabolic regulation, which are poorly understood. To achieve the aim, we examined the impact and mechanism of heat events on S. frugiperda by using multiple approaches: ecological measurements, transcriptomics, metabolomics, RNAi, and CRISPR/Cas9 technology. We observed that several physiological indices (larval survival rate, larval period, pupation rate, pupal weight, eclosion rate, and average fecundity) decreased as the temperature increased, with the 32 °C treatment displaying a significant difference from the control group at 26 °C. Significantly upregulated expression of genes encoding endochitinase and chitin deacetylase was observed in the chitin-binding, extracellular region, and carbohydrate metabolic process GO terms of hemolymph, fat body, and brain, exhibiting a tissue-specific pattern. Significantly enriched pathways (e.g., cutin, suberin, and wax biosynthesis; oxidative phosphorylation and cofactor biosynthesis; diverse amino acid biosynthesis and degradation; carbon metabolism; and energy metabolism), all of which are essential for S. frugiperda larvae to tolerate temperature, were found in metabolites that were expressed differently. Successful RNA interference targeting of the three chitin-related genes reduced gene expression levels and larval survival rate. Knockout of the endochitinase gene by using the CRISPR/Cas9 system significantly reduced the relative gene expression and increased sensitivity to high-temperature exposure. On the basis of our findings, theoretical foundations for understanding the high-temperature tolerance of S. frugiperda populations and latent genetic control strategies were established.

Biotransformation of insect processing residues: Production of lactic acid bacterial biomass and associated partial removal of proteins from chitin.

Processing of edible insects typically involves fractionating into high-value food ingredients, which results in by-products containing chitin and insoluble proteins. This study examined the effectiveness of lactic acid bacteria (LAB) in removing proteins from chitin in insect processing residues. Lesser mealworm processing residues were biologically treated for 48 and 120 h using LAB strains without added carbon sources. Results showed partial deproteinization, up to 29 % with Levilactobacillus brevis after 120 h. Most LAB grew up to 2 log10 colony-forming units/mL in the first 48 h. Confocal microscopy and Fourier-transform infrared spectra indicated that some protein remained attached to chitin. The molecular weight of solubilized proteins was affected by strain and time of incubation, with antioxidant activity increasing significantly after 120 h with Lacticaseibacillus paracasei. The biological treatment of insect processing streams can be a sustainable approach to producing high amounts of LAB biomass with subsequent protein solubilization and chitin release.

Display of Bacterial Exochitanase on Bacillus subtilis Spores Improved Enzyme Stability and Recyclability.

Chitin is the second most prevalent polysaccharide found in nature, following cellulose. Amino-oligosaccharides, the byproducts of chitin degradation, exhibit favorable biological properties and potential for various uses. Chitinases play a crucial function in the breakdown of chitin, and their exceptionally effective production has garnered significant interest. Here, in this study, the exochitinase PbChiA, obtained from Paenibacillus barengoltzii, was recombinantly produced and immobilized using the CotG surface protein of Bacillus subtilis WB800N. The resulting strain Bacillus subtilis WB800N pHS-CotG-Chi exhibited exceptional heat stability and efficacy across various pH levels. The chitinolytic activity of the enzyme, which had been isolated and immobilized on the spore surface, was measured to be approximately 16.06 U/mL. Including Ni2+, Zn+2, and K+, and EDTA at various concentration levels in the reaction system, has significantly enhanced the activity of the immobilized enzyme. The immobilized exochitinase demonstrated a notable rate of recycling, as the recombinant spores sustained a relative enzyme activity of more than 70% after three cycles and 62.7% after four cycles. These findings established a basis for additional investigation into the role and practical use of the immobilized bacterial exochitinase in industry.

Development of a Chitin-Based Purification System Utilizing Chitin-Binding Domain and Tobacco Etch Virus Protease Cleavage for Efficient Recombinant Protein Recovery.

This study aims to develop an efficient chitin-based purification system, leveraging a novel design where the target proteins, superfolding green fluorescent protein (sfGFP) and Thermus antranikianii trehalose synthase (TaTS), fused with a chitin-binding domain (ChBD) from Bacillus circulans WL-12 chitinase A1 and a tobacco etch virus protease (TEVp) cleavage site. This configuration allows for the effective immobilization of the target proteins on chitin beads, facilitating the removal of endogenous proteins. A mutant TEVp, H-TEVS219V-ChBD, fused with the His-tag and ChBD, is employed to cleave the target proteins from the chitin beads specifically. Subsequently, fresh chitin beads are added for adsorption to remove H-TEVS219V-ChBD in the solution, thereby significantly improving the purity of the target protein. Our results confirm that this system can efficiently and specifically purify and recover sfGFP and TaTS, achieving electrophoretic-grade purity exceeding 90%. This system holds significant potential for industrial production and other applications.

Isolation and characterization of Saccharomyces cerevisiae mutants with increased cell wall chitin using fluorescence-activated cell sorting.

Yeast cell wall chitin has been shown to bind grape pathogenesis-related chitinases that are the primary cause of protein haze in wines, suggesting that yeast cell walls may be applied for haze protection. Here, we present a high-throughput screen to identify yeast strains with high cell wall chitin using a reiterative enrichment strategy and fluorescence-activated cell sorting of cells labelled with either GFP-tagged chitinase or Calcofluor white. To assess the validity of the strategy, we first used a pooled deletion strain library of Saccharomyces cerevisiae. The strategy enriched for deletion mutants with genes that had previously been described as having an impact on chitin levels. Genes that had not previously been linked to chitin biosynthesis or deposition were also identified. These genes are involved in cell wall maintenance and/or membrane trafficking functions. The strategy was then applied to a mutagenized population of a commercial wine yeast strain, S. cerevisiae EC1118. Enriched mutant strains showed significantly higher cell wall chitin than the wild type and significantly reduced the activity of chitinases in synthetic model wine, suggesting that these strains may be able to reduce haze formation in wine.

Nanomaterial-Encapsulated dsRNA-Targeting Chitin Pathway─A Potential Efficient and Eco-Friendly Strategy against Cotton Aphid, Aphis gossypii (Hemiptera: Aphididae).

The r-strategy pests are very challenging to effectively control because of their rapid population growth and strong resurgence potential and are more prone to developing pesticide resistance. As a typical r-strategy pest, the cosmopolitan cotton aphid, Aphis gossypii Glover, seriously impacts the growth and production of cucurbits and cotton. The present study developed a SPc/double-stranded RNA (dsRNA)/botanical strategy to enhance the control efficacy of A. gossypii. The results demonstrated that the expression of two chitin pathway genes AgCHS2 and AgHK2 notably changed in A. gossypii after treated by three botanical pesticides, 1% azadirachtin, 1% matrine, and 5% eucalyptol. SPc nanocarrier could significantly enhance the environmental stability, cuticle penetration, and interference efficiency of dsRNA products. The SPc/dsRNA/botanical complex could obviously increase the mortality of A. gossypii in both laboratory and greenhouse conditions. This study provides an eco-friendly control technique for enhanced mortality of A. gossypii and lower application of chemical pesticides. Given the conservative feature of chitin pathway genes, this strategy would also shed light on the promotion of management strategies against other r-strategy pests using dsRNA/botanical complex nanopesticides.

Genomics, Proteomics, and Antifungal Activity of Chitinase from the Antarctic Marine Bacterium Curtobacterium sp. CBMAI 2942.

This study aimed to evaluate the genomic profile of the Antarctic marine Curtobacterium sp. CBMAI 2942, as well as to optimize the conditions for chitinase production and antifungal potential for biological control. Assembly and annotation of the genome confirmed the genomic potential for chitinase synthesis, revealing two ChBDs of chitin binding (Chi C). The optimization enzyme production using an experimental design resulted in a 3.7-fold increase in chitinase production. The chitinase enzyme was identified by SDS-PAGE and confirmed through mass spectrometry analysis. The enzymatic extract obtained using acetone showed antifungal activity against the phytopathogenic fungus Aspergillus sp. series Nigri CBMAI 1846. The genetic capability of Curtobacterium sp. CBMAI 2942 for chitin degradation was confirmed through genomic analysis. The basal culture medium was adjusted, and the chitinase produced by this isolate from Antarctica showed significant inhibition against Aspergillus sp. Nigri series CBMAI 1846, which is a tomato phytopathogenic fungus. This suggests that this marine bacterium could potentially be used as a biological control of agricultural pests.

Molecular architecture of chitin and chitosan-dominated cell walls in zygomycetous fungal pathogens by solid-state NMR.

Zygomycetous fungal infections pose an emerging medical threat among individuals with compromised immunity and metabolic abnormalities. Our pathophysiological understanding of these infections, particularly the role of fungal cell walls in growth and immune response, remains limited. Here we conducted multidimensional solid-state NMR analysis to examine cell walls in five Mucorales species, including key mucormycosis causative agents like Rhizopus and Mucor species. We show that the rigid core of the cell wall primarily comprises highly polymorphic chitin and chitosan, with minimal quantities of ß-glucans linked to a specific chitin subtype. Chitosan emerges as a pivotal molecule preserving hydration and dynamics. Some proteins are entrapped within this semi-crystalline chitin/chitosan layer, stabilized by the sidechains of hydrophobic amino acid residues, and situated distantly from ß-glucans. The mobile domain contains galactan- and mannan-based polysaccharides, along with polymeric α-fucoses. Treatment with the chitin synthase inhibitor nikkomycin removes the ß-glucan-chitin/chitosan complex, leaving the other chitin and chitosan allomorphs untouched while simultaneously thickening and rigidifying the cell wall. These findings shed light on the organization of Mucorales cell walls and emphasize the necessity for a deeper understanding of the diverse families of chitin synthases and deacetylases as potential targets for novel antifungal therapies.

Enhanced degradation of insoluble chitin: Engineering high-efficiency chitinase fusion enzymes for sustainable applications.

N-acetyl-D-glucosamine and its dimer are degradation products of chitin waste with great potential in therapeutic and agricultural applications. However, the hydrolysis of insoluble chitin by chitinases remains a major bottleneck. This study investigated the biochemical properties and catalytic mechanisms of PoChi chitinase obtained from Penicillium oxalicum with a focus on enhancing its efficiency during the degradation of insoluble chitin. Recombinant plasmids were engineered to incorporate chitin-binding (ChBD) and/or fibronectin III (FnIII) domains. Notably, PoChi-FnIII-ChBD exhibited the highest substrate affinity (Km = 2.7 mg/mL) and a specific activity of 15.4 U/mg, which surpasses those of previously reported chitinases. These findings highlight the potential of engineered chitinases in advancing industrial biotechnology applications and offer a promising approach to more sustainable chitin waste management.

Heterologous expression, characterisation and 3D-structural insights of GH18 chitinases derived from sago palm (Metroxylon sagu).

Although plants don't have chitins, they produce chitinases to protect themselves from biotic and abiotic stressors. There are two forms of chitinases found in organisms: glycosyl hydrolase 18 (GH18) and 19 (GH19) families. Plant GH19 chitinases are well known for their role in protecting against pathogens, but the roles of GH18 chitinases have not been fully elucidated. This study aimed to produce and characterise two recombinant GH18 chitinases from Metroxylon sagu. Two GH18 chitinase genes, MsChi1 and MsChi2, were identified, with nucleotide sequences of 1009 and 1308 bp, respectively. The proteins encoded by MsChi1 and MsChi2 genes were single polypeptide chains of 310 and 300 amino acids with predicted molecular masses of 31.21 and 30.15 kDa, respectively. Both cDNAs were cloned and expressed in the GS115 strain of Pichia pastoris. Recombinant MsChi1 and MsChi2 exhibited optimal activity at 60 °C with acidic pH 4.0 and 5.0, respectively. Both recombinant enzymes could hydrolyze synthetic and natural substrates (colloidal chitin). rMsChi1 preferred 4-nitrophenol N,N'-diacetyl-ß-D chitobioside, while rMsChi2 preferred 4-nitrophenol N,N',N″-triacetyl-ß-D chitotriose, suggesting they might function as exochitinase and endochitinase, respectively. They also demonstrated antifungal activities against tested fungi. Homology modeling indicated ASP and GLU as essential residues for proton donation and acceptance.

Overexpression of plant chitin receptors in wheat confers broad-spectrum resistance to fungal diseases.

Wheat (Triticum aestivum L.) is a globally staple crop vulnerable to various fungal diseases, significantly impacting its yield. Plant cell surface receptors play a crucial role in recognizing pathogen-associated molecular patterns (PAMPs) and activating PAMP-triggered immunity, boosting resistance against a wide range of plant diseases. Although the role of plant chitin receptor CERK1 in immune recognition and defense has been established in Arabidopsis and rice, its function and potential agricultural applications in enhancing resistance to crop diseases remain largely unexplored. Here, we identify and characterize TaCERK1 in Triticeae crop wheat, uncovering its involvement in chitin recognition, immune regulation, and resistance to fungal diseases. By a comparative analysis of CERK1 homologs in Arabidopsis and monocot crops, we demonstrate that AtCERK1 in Arabidopsis elicits the most robust immune response. Moreover, we show that overexpressing TaCERK1 and AtCERK1 in wheat confers resistance to multiple fungal diseases, including Fusarium head blight, stripe rust, and powdery mildew. Notably, transgenic wheat lines with moderately expressed AtCERK1 display superior disease resistance and heightened immune responses without adversely affecting growth and yield, compared to TaCERK1 overexpression transgenics. Our findings highlight the significance of plant chitin receptors across diverse plant species and suggest potential strategies for bolstering crop resistance against broad-spectrum diseases in agricultural production through the utilization of plant immune receptors.

Disruption to de novo uridine biosynthesis alters ß-1,3-glucan masking in Candida albicans.

The uridine derivatives UDP-glucose and UDP-N-acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of ß-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing ß-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of ß-1,3-glucan. Moreover, the ura3ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCECandida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly ß-1,3-glucan. However, ß-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase ß-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors.

Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII.

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation has been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.