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Pruritus is the leading symptom of dermatophytosis. Microsporium canis is one of the predominant dermatophytes causing dermatophytosis. However, the pruritogenic agents and the related molecular mechanisms of the dermatophyte M canis remain poorly understood. In this study, the secretion of the dermatophyte M canis was found to dose-dependently evoke itch in mice. The fungal peptide micasin secreted from M canis was then identified to elicit mouse significant scratching and itching responses. The peptide micasin was further revealed to directly activate mouse dorsal root ganglia neurons to mediate the nonhistaminergic itch. Knockout and antagonistic experiments demonstrated that MRGPRX1/C11/A1 rather than MRGPRX2/b2 activated by micasin contributed to pruritus. The chimeras and single-amino acid variants of MRGPRX1 showed that 3 domains (extracellular loop 3, transmembrane helical domain 3, and transmembrane helical domain 6) and 4 hydrophobic residues (Y99, F237, L240, and W241) of MRGPRX1 played the key role in micasin-triggered MRGPRX1 activation. Our study sheds light on the dermatophytosis-associated pruritus and may provide potential therapeutic targets and strategies against pruritus caused by dermatophytes.

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Introduction: Invasive fungal infections (IFIs) are fatally threatening to critical patients. The fungal defensin as an antifungal protein can widely inhibit fungi. Methods: In this study, eight antifungal genes from different filamentous fungi were optimized by synonymous codon bias and heterologously expressed in Escherichia coli. Results and discussion: Only the antifungal protein (AFP) from Aspergillus giganteus was produced, whereas the AFP from its mutation of the chitin-binding domain could not be expressed, thereby suggesting the importance of the motif for protein folding. In addition, the recombinant AFP (rAFP, 100 µg/mL) pre-heated at 50°C for 1 h effectively inhibited Paecilomyces variotii CICC40716 of IFIs by 55%, and no cell cytotoxicity was observed in RAW264.7 cells. After being pre-heated at 50°C for 8 h, the fluorescence emission intensity of the rAFP decreased and shifted from 343 nm to 335 nm. Moreover, the helix and ß-turn of the rAFP gradually decreased with the pre-heated treatment temperature of 50°C via circular dichroism spectroscopy. Propidium iodide staining revealed that the rAFP could cause damage to the cell membrane. Moreover, the corresponding differentially expressed genes (DEGs) for downregulation such as amino sugar and nucleotide sugar metabolism, as well as mitogen-activated protein kinase (MAPK) signaling pathway involved in the cell wall integrity were found via the RNA-seq of rAFP treatment. By contrast, the upregulated DEGs were enriched in response to the oxidative stress of Biological Process by the Gene Ontology (GO) database. The encoding proteins of laccase, multicopper oxidase, and nitroreductase that contributed to reactive oxygen species (ROS) scavenging could be recognized. These results suggested that the rAFP may affect the integrity of the cell wall and cell membrane, and promote the increase in ROS, thereby resulting in fungal death. Consequently, drug development could be based on the inhibitory effect of the rAFP on IFIs.

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Pichia pastoris is the widely used expression system for producing recombinant secretory proteins. It is known that Kex2 protease plays a vital role in the process of protein secretion, in which the P1' site affects its cleavage efficiency. To enhance the expression level of fungal defensin-derived peptide NZ2114, this work attempts to optimize the P1' site of Kex2 by replacing it with 20 amino acids in turn. The results showed that when the amino acid of the P1' site was changed to Phe (F), the yield of target peptide significantly increased from 2.39 g/L to 4.81 g/L. Additionally, the novel peptide F-NZ2114 (short for FNZ) showed strong antimicrobial activity against Gram-positive (G+) bacteria, especially for Staphylococcus aureus and Streptococcus agalactiae (MIC: 4-8 µg/mL). The FNZ was very stable and retained high activity in various conditions; in addition, a low cytotoxicity and no hemolysis were observed even at a high concentration of 128 µg/mL, and a longer postantibiotic effect was reached. The above results indicate that this engineering strategy provided a feasible optimization scheme for enhancing the expression level and druggability of this antimicrobial peptide from fungal defensin and other similar targets by this updated recombinant yeast.

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Staphylococcus aureus (S. aureus) is one of the most common pathogenic bacteria responsible for causing a life-threatening peritonitis disease. NZX, as a variant of fungal defensin plectasin, displayed potent antibacterial activity against S. aureus. In this study, the antibacterial and resistance characteristics, pharmacokinetics, and pharmacodynamics of NZX against the S. aureus E48 and S. aureus E48-induced mouse peritonitis model were studied, respectively. NZX exhibited a more rapid killing activity to S. aureus (minimal inhibitory concentration, 1 µg/ml) compared with linezolid, ampicillin and daptomycin, and serial passaging of S. aureus E48 for 30 days at 1/2 × MIC, NZX had a lower risk of resistance compared with ampicillin and daptomycin. Also, it displayed a high biocompatibility and tolerance to physiological salt, serum environment, and phagolysosome proteinase environment, except for acid environment in phagolysosome. The murine serum protein-binding rate of NZX was 89.25% measured by ultrafiltration method. Based on the free NZX concentration in serum after tail vein administration, the main pharmacokinetic parameters for T1/2, Cmax, Vd, MRT, and AUC ranged from 0.32 to 0.45 h, 2.85 to 20.55 µg/ml, 1469.10 to 2073.90 ml/kg, 0.32 to 0.56 h, and 1.11 to 8.89 µg.h/ml, respectively. Additionally, the in vivo pharmacodynamics against S. aureus demonstrated that NZX administrated two times by tail vein at 20 mg/kg could rescue all infected mice in the lethal mouse peritonitis model. And NZX treatment (20 mg/kg) significantly reduced CFU counts in the liver, lung, and spleen, especially for intracellular bacteria in the peritoneal fluid, which were similar or superior to those of daptomycin. In vivo efficacies of NZX against total bacteria and intracellular bacteria were significantly correlated with three PK/PD indices of ƒAUC/MIC, ƒCmax/MIC, and ƒT% > MIC analyzed by a sigmoid maximum-effect model. These results showed that NZX may be a potential candidate for treating peritonitis disease caused by intracellular S. aureus.

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Staphylococcus hyicus is recognized as a leading pathogen of exudative epidermitis in modern swine industry. Antimicrobial peptides are attractive candidates for development as potential therapeutics to combat the serious threats of the resistance of S. hyicus. In this study, a series of derivatives were designed based on the NZ2114 template with the aim of obtaining peptides with more potent antimicrobial activity through changing net positive charge or hydrophobicity. Among them, a variant designated as NZL was highly expressed in Pichia pastoris (P. pastoris) with total secreted protein of 1505 mg/L in a 5-L fermenter and exhibited enhanced antimicrobial activity relative to parent peptide NZ2114. Additionally, NZL could kill over 99% of S. hyicus NCTC10350 in vitro within 8 h and in Hacat cells. The results of membrane permeabilization assay, morphological observations, peptide localization assay showed that NZL had potent activity against S. hyicus, which maybe kill S. hyicus through action on the cell wall. NZL also showed an effective therapy in a mouse peritonitis model caused by S. hyicus, superior to NZ2114 or ceftriaxone. Overall, these findings can contribute to explore a novel potential candidate against S. hyicus infections.

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BACKGROUND: "Invertebrate defensins" belong to the cysteine-stabilized alpha-beta (CS-αß), also known as the scorpion toxin-like, superfamily. Some other peptides belonging to this superfamily of defensive peptides are indistinguishable from "defensins," but have been assigned other names, making it unclear what, if any, criteria must be met to qualify as an "invertebrate defensin." In addition, there are other groups of defensins in invertebrates and vertebrates that are considered to be evolutionarily unrelated to those in the CS-αß superfamily. This complicates analyses and discussions of this peptide group. This paper investigates the criteria for classifying a peptide as an invertebrate defensin, suggests a reference cysteine array that may be helpful in discussing peptides in this superfamily, and proposes that the superfamily (rather than the name "defensin") is the appropriate context for studying the evolution of invertebrate defensins with the CS-αß fold. METHODS: CS-αß superfamily sequences were identified from previous literature and BLAST searches of public databases. Sequences were retrieved from databases, and the relevant motifs were identified and used to create a conceptual alignment to a ten-cysteine reference array. Amino acid sequences were aligned in MEGA6 with manual adjustments to ensure accurate alignment of cysteines. Phylogenetic analyses were performed in MEGA6 (maximum likelihood) and MrBayes (Bayesian). RESULTS: Across invertebrate taxa, the term "defensin" is not consistently applied based on number of cysteines, cysteine spacing pattern, spectrum of antimicrobial activity, or phylogenetic relationship. The analyses failed to reveal any criteria that unify "invertebrate defensins" and differentiate them from other defensive peptides in the CS-αß superfamily. Sequences from various groups within the CS-αß superfamily of defensive peptides can be described by a ten-cysteine reference array that aligns their defining structural motifs. CONCLUSIONS: The proposed ten-cysteine reference array can be used in addition to current nomenclature to compare sequences in the CS-αß superfamily and clarify their features relative to one another. This will facilitate analysis and discussion of "invertebrate defensins" in an appropriate evolutionary context, rather than relying on nomenclature.

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