Function and Mechanism of Antiviral Wasp Venom Peptide Protopolybia-MP III and Its Derivatives against HSV-1.

Viruses are one of the leading causes of human disease, and many highly pathogenic viruses still have no specific treatment drugs. Therefore, producing new antiviral drugs is an urgent matter. In our study, we first found that the natural wasp venom peptide Protopolybia-MP III had a significant inhibitory effect on herpes simplex virus type 1 (HSV-1) replication in vitro by using quantitative real-time PCR (qPCR), Western blotting, and plaque-forming assays. Immunofluorescence analysis showed Protopolybia-MP III could enter cells, and it inhibited multiple stages of the HSV-1 life cycle, including the attachment, entry/fusion, and post-entry stages. Furthermore, ultracentrifugation and electron microscopy detected that Protopolybia-MP III significantly suppressed HSV-1 virion infectivity at different temperatures by destroying the integrity of the HSV-1 virion. Finally, by comparing the antiviral activity of Protopolybia-MP III and its mutants, a series of peptides with better anti-HSV-1 activity were identified. Overall, this work found the function and mechanism of the antiviral wasp venom peptide Protopolybia-MP III and its derivatives against HSV-1 and laid the foundation for the research and development of wasp venom-derived antiviral candidate peptide drugs.

Cationicity Enhancement on the Hydrophilic Face of Ctriporin Significantly Reduces Its Hemolytic Activity and Improves the Antimicrobial Activity against Antibiotic-Resistant ESKAPE Pathogens.

The ESKAPE pathogen-associated antimicrobial resistance is a global public health issue, and novel therapeutic strategies are urgently needed. The short cationic antimicrobial peptide (AMP) family represents an important subfamily of scorpion-derived AMPs, but high hemolysis and poor antimicrobial activity hinder their therapeutic application. Here, we recomposed the hydrophilic face of Ctriporin through lysine substitution. We observed non-linear correlations between the physiochemical properties of the peptides and their activities, and significant deviations regarding the changes of antimicrobial activities against different bacterial species, as well as hemolytic activity. Most importantly, we obtained two Ctriporin analogs, CM5 and CM6, these two have significantly reduced hemolytic activity and more potent antimicrobial activities against all tested antibiotic-resistant ESKAPE pathogens. Fluorescence experiments indicated they may perform the bactericidal function through a membrane-lytic action model. Our work sheds light on the potential of CM5 and CM6 in developing novel antimicrobials and gives clues for optimizing peptides from the short cationic AMP family.

Characterization of the Hemolytic Activity of Mastoparan Family Peptides from Wasp Venoms.

Biologically active peptides have attracted increasing attention in research on the development of new drugs. Mastoparans, a group of wasp venom linear cationic α-helical peptides, have a variety of biological effects, including mast cell degranulation, activation of protein G, and antimicrobial and anticancer activities. However, the potential hemolytic activity of cationic α-helical peptides greatly limits the clinical applications of mastoparans. Here, we systematically and comprehensively studied the hemolytic activity of mastoparans based on our wasp venom mastoparan family peptide library. The results showed that among 55 mastoparans, 18 had strong hemolytic activity (EC50 ≤ 100 µM), 14 had modest hemolytic activity (100 µM < EC50 ≤ 400 µM) and 23 had little hemolytic activity (EC50 > 400 µM), suggesting functional variation in the molecular diversity of mastoparan family peptides from wasp venom. Based on these data, structure-function relationships were further explored, and, hydrophobicity, but not net charge and amphiphilicity, was found to play a critical role in the hemolytic activity of mastoparans. Combining the reported antimicrobial activity with the present hemolytic activity data, we found that four mastoparan peptides, Parapolybia-MP, Mastoparan-like peptide 12b, Dominulin A and Dominulin B, have promise for applications because of their high antimicrobial activity (MIC ≤ 10 µM) and low hemolytic activity (EC50 ≥ 400 µM). Our research not only identified new leads for the antimicrobial application of mastoparans but also provided a large chemical space to support the molecular design and optimization of mastoparan family peptides with low hemolytic activity regardless of net charge or amphiphilicity.

Characterization of the Molecular Diversity and Degranulation Activity of Mastoparan Family Peptides from Wasp Venoms.

Wasp stings have become an increasingly serious public health problem because of their high incidence and mortality rates in various countries and regions. Mastoparan family peptides are the most abundant natural peptides in hornet venoms and solitary wasp venom. However, there is a lack of systematic and comprehensive studies on mastoparan family peptides from wasp venoms. In our study, for the first time, we evaluated the molecular diversity of 55 wasp mastoparan family peptides from wasp venoms and divided them into four major subfamilies. Then, we established a wasp peptide library containing all 55 known mastoparan family peptides by chemical synthesis and C-terminal amidation modification, and we systematically evaluated their degranulation activities in two mast cell lines, namely the RBL-2H3 and P815 cell lines. The results showed that among the 55 mastoparans, 35 mastoparans could significantly induce mast cell degranulation, 7 mastoparans had modest mast cell degranulation activity, and 13 mastoparans had little mast cell degranulation activity, suggesting functional variation in mastoparan family peptides from wasp venoms. Structure-function relationship studies found that the composition of amino acids in the hydrophobic face and amidation in the C-terminal region are critical for the degranulation activity of mastoparan family peptides from wasp venoms. Our research will lay a theoretical foundation for studying the mechanism underlying the degranulation activity of wasp mastoparans and provide new evidence to support the molecular design and molecular optimization of natural mastoparan peptides from wasp venoms in the future.

Schixator, a new FXa inhibitor from Schistosoma japonicum with antithrombotic effect and low bleeding risk.

Schistosoma japonicum is a parasitic worm that lives in the mesenteric vein of its host and feeds on blood, suggesting that it might be a natural resource of novel anticoagulants. Here, by comprehensive analyses of the genomic sequences of Schistosoma japonicum, a new Kunitz-type gene precursor was identified. The Kunitz-type gene precursor codes for an 18-residue signal peptide and a 60-residue mature peptide. The Kunitz peptide was functionally expressed, and it had apparent inhibitory activity towards the intrinsic coagulation pathway but no effect on the extrinsic coagulation pathway even at the high concentration of 3 µM. Enzyme and inhibitor experiments further showed that the Kunitz domain peptide was a potent and selective FXa inhibitor, so it was named Schixator (Schistosoma FXa inhibitor). Schixator inhibits coagulation factor FXa with a Ki of 2.66 nM, but had weak inhibitory activity towards chymotrypsin, FXIa, plasma kallikrein, and plasmin, and no inhibitory activity towards trypsin, elastase, FIIa or FXIIa. In vivo, the intravenous administration of Schixator into mice dramatically decreased the number of thrombi in the carotid artery in an FeCl3-induced thrombus formation model without producing bleeding complications. To the best of our knowledge, Schixator is the first potent and selective FXa inhibitor from parasitic worms with antithrombotic effects and a low bleeding risk that provides a new clue for lead drug discovery against thrombosis-associated human diseases.

Corrigendum: Fine-Tuning of Alkaline Residues on the Hydrophilic Face Provides a Non-toxic Cationic α-Helical Antimicrobial Peptide Against Antibiotic-Resistant ESKAPE Pathogens.

[This corrects the article DOI: 10.3389/fmicb.2021.684591.].

Fine-Tuning of Alkaline Residues on the Hydrophilic Face Provides a Non-toxic Cationic α-Helical Antimicrobial Peptide Against Antibiotic-Resistant ESKAPE Pathogens.

Antibiotic-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has become a serious threat to public health worldwide. Cationic α-helical antimicrobial peptides (CαAMPs) have attracted much attention as promising solutions in post-antibiotic era. However, strong hemolytic activity and in vivo inefficacy have hindered their pharmaceutical development. Here, we attempt to address these obstacles by investigating BmKn2 and BmKn2-7, two scorpion-derived CαAMPs with the same hydrophobic face and a distinct hydrophilic face. Through structural comparison, mutant design and functional analyses, we found that while keeping the hydrophobic face unchanged, increasing the number of alkaline residues (i.e., Lys + Arg residues) on the hydrophilic face of BmKn2 reduces the hemolytic activity and broadens the antimicrobial spectrum. Strikingly, when keeping the total number of alkaline residues constant, increasing the number of Lys residues on the hydrophilic face of BmKn2-7 significantly reduces the hemolytic activity but does not influence the antimicrobial activity. BmKn2-7K, a mutant of BmKn2-7 in which all of the Arg residues on the hydrophilic face were replaced with Lys, showed the lowest hemolytic activity and potent antimicrobial activity against antibiotic-resistant ESKAPE pathogens. Moreover, in vivo experiments indicate that BmKn2-7K displays potent antimicrobial efficacy against both the penicillin-resistant S. aureus and the carbapenem- and multidrug-resistant A. baumannii, and is non-toxic at the antimicrobial dosages. Taken together, our work highlights the significant functional disparity of Lys vs Arg in the scorpion-derived antimicrobial peptide BmKn2-7, and provides a promising lead molecule for drug development against ESKAPE pathogens.

An Smp43-Derived Short-Chain α-Helical Peptide Displays a Unique Sequence and Possesses Antimicrobial Activity against Both Gram-Positive and Gram-Negative Bacteria.

Scorpion venoms are rich resources of antimicrobial peptides (AMPs). While the short-chain noncysteine-containing AMPs have attracted much attention as templates for drug development, the antimicrobial potential of long-chain noncysteine-containing AMPs has been largely overlooked. Here, by using the online HeliQuest server, we designed and analyzed a series of 14-residue fragments of Smp43, a 43-residue long-chain noncysteine-containing AMP identified from the venom of Scorpio maurus palmatus. We found that Smp43(1-14) shows high antimicrobial activity against both Gram-positive and Gram-negative bacteria and is nontoxic to mammalian cells at the antimicrobial dosage. Sequence alignments showed that the designed Smp43(1-14) displays a unique primary structure that is different from other natural short-chain noncysteine-containing AMPs from scorpions, such as Uy17, Uy192 and IsCT. Moreover, the peptide Smp43(1-14) caused concentration-dependent fluorescence increases in the bacteria for all of the tested dyes, propidium iodide, SYTOXTM Green and DiSC3-5, suggesting that the peptide may kill the bacteria through the formation of pore structures in the plasma membrane. Taken together, our work sheds light on a new avenue for the design of novel short-chain noncysteine-containing AMPs and provides a good peptide template with a unique sequence for the development of novel drugs for use against bacterial infectious diseases.

Identification of the scorpion venom-derived antimicrobial peptide Hp1404 as a new antimicrobial agent against carbapenem-resistant Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is becoming a troublesome issue worldwide, and anti-CRAB drug research and development is urgently needed. To identify new anti-CRAB drug leads, we investigated seven scorpion venom-derived α-helical peptides that differ in their sequence composition and length. Three peptides, Hp1404, ctriporin and Im5, showed antimicrobial activities against Acinetobacter baumannii. Further antimicrobial assays revealed that Hp1404 exhibited the best cell selectivity with high anti-CRAB and low hemolytic activities. Fluorescence assays demonstrated that Hp1404 can induce dose-dependent disruptions of the bacterial cell membrane, implying a membrane-lytic mode of action. Taken together, our work sheds light on the potential of the scorpion venom-derived peptide Hp1404 for the development of novel antimicrobial agents against CRAB infections.

Two new cationic α-helical peptides identified from the venom gland of Liocheles australasiae possess antimicrobial activity against methicillin-resistant staphylococci.

Methicillin-resistant staphylococci have become growing threats to human health, and novel antimicrobials are urgently needed. Natural antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics. Here, two novel cationic α-helical antimicrobial peptides, Lausporin-1 and Lausporin-2, were identified from the venom gland of the scorpion L. australasiae through a cDNA library screening strategy. Biochemical analyses demonstrated that Lausporin-1 and Lausporin-2 are cationic α-helical amphipathic molecules. Antimicrobial assays demonstrated that the two peptides possess antibacterial activities against several species of antibiotic-resistant staphylococci. Importantly, they are active against methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus capitis, with the minimum inhibitory concentrations ranging from 2.5 to 10 µg/ml. Moreover, both peptides can induce dose-dependent plasma membrane disruptions of the bacteria. In short, our work expands the knowledge of the scorpion L. australasiae venom-derived AMPs and sheds light on the potential of Lausporin-1 and Lausporin-2 in the development of novel drugs against methicillin-resistant staphylococci.

Synergistic effects of AAGL and anti-PD-1 on hepatocellular carcinoma through lymphocyte recruitment to the liver.

OBJECTIVE: Therapy for hepatocellular carcinoma (HCC) is a major challenge, and targeted therapies provide only a modest benefit in terms of overall survival. Treatment with antibodies to programmed cell death protein 1 (PD-1)/PD-L1 can restore the functions of tumor-infiltrating T cells in HCC and has shown clinical efficacy in 20% of patients with advanced HCC. Novel approaches are urgently needed to treat HCC and to augment the efficacy of immunotherapy. METHODS: Tumor-bearing mice were treated with Agrocybe aegerita galectin (AAGL) alone or in combination with anti-PD-1, and the tumor sizes and lifespans of mice were determined. Transcriptome analysis, cytokine analysis, flow cytometry analysis of the number and proportion of immune cell subsets in the liver and spleen, and molecular and cellular analyses of tumors were used to define the underlying mechanisms. RESULTS: AAGL significantly inhibited the growth of liver tumors in a dose-dependent manner. Furthermore, AAGL increased the expression of multiple cytokines and chemokines in tumor-bearing mouse livers; this effect was associated with the activation and migration of T cells and macrophages, in agreement with the in vitro results. Importantly, the aggregation of T cells and macrophages induced by AAGL in tumor-bearing mouse livers clearly enhanced the response to PD-1 blockade immunotherapy. CONCLUSIONS: The results showed that AAGL induced the activation and migration of lymphocytes to the liver, and that the combination of AAGL and anti-PD-1 may be a promising strategy for HCC treatment.

Cloning and identification of a new multifunctional Ascaris-type peptide from the hemolymph of Buthus martensii Karsch.

Only a few work have been done for peptides from non-venom gland tissues of venomous animals. Here, with the help of the whole body transcriptomic and the hemolymph proteomic data of the Chinese scorpion Buthus martensii Karsch, we identified the first Ascaris-type peptide BmHDP from scorpion hemolymph. The precursor of BmHDP has 80 residues, including a 16 residue signal peptide and a 64 residue mature peptide. The mature peptide has 10 conserved cysteines and adopts a conserved Ascaris-type fold. Using combined inclusion body refolding and biochemical identification strategies, recombinant BmHDP was obtained successfully. Protease inhibitory assays showed that BmHDP inhibited chymotrypsin apparently at a concentration of 8 nM. Patch-clamp experiments showed that BmHDP inhibited the Kv1.3 potassium channel apparently at a concentration of 1000 nM. Coagulation experiment assays showed that BmHDP inhibited intrinsic coagulation pathway apparently at a concentration of 500 nM. To the best of our knowledge, BmHDP is the first Ascaris-type peptide from scorpion hemolymph. Our work highlighted a functional link between scorpion non-venom gland peptides and venom gland toxin peptides, and suggested that scorpion hemolymph might be a new source of bioactive peptides.

A novel dry-blending method to reduce the coefficient of thermal expansion of polymer templates for OTFT electrodes.

Among the patterning technologies for organic thin-film transistors (OTFTs), the fabrication of OTFT electrodes using polymer templates has attracted much attention. However, deviations in the electrode alignment occur because the coefficient of thermal expansion (CTE) of the polymer template is much higher than the CTE of the dielectric layer. Here, a novel dry-blending method is described in which SiO2 nanoparticles are filled into a grooved silicon template, followed by permeation of polydimethylsiloxane (PDMS) into the SiO2 nanoparticle gaps. The SiO2 nanoparticles in the groove are extracted by curing and peeling off PDMS to prepare a PDMS/SiO2 composite template with a nanoparticle content of 83.8 wt %. The composite template has a CTE of 96 ppm/°C, which is a reduction by 69.23% compared with the original PDMS template. Finally, we achieved the alignment of OTFT electrodes using the composite template.

Dramatically changed immune-related molecules as early diagnostic biomarkers of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the main type of lung cancer, with a low 5-year survival rate because of the absence of effective clinical biomarkers for early diagnosis. Based on the immunosurveillance theory, we proposed that changes in the immune system are more pronounced than tumour-associated antigens during the early stage of cancer. Therefore, a new strategy was designed to screen early diagnostic biomarkers from peripheral leukocytes in early-stage NSCLCs with transcriptome sequencing. A total of 358 immune-related differentially expressed genes were identified between early-NSCLC patients and healthy individuals. Orosomucoid-1 (ORM1, a acute phase protein), the total ORM and chitotriosidase-1 (involved in degradation of chitobiose) were selected for further verification in 210 serum samples by western blotting, ELISA and nephelometry immunoassay (based on immuno-scatter turbidmetry). Receiver operating characteristic curve analysis show that ORM1 and total ORM have excellent diagnostic efficacies, with area under the curve of 0.862 and 0.920, respectively, which significantly distinguished very early-NSCLC (IA) from healthy samples. Flow cytometry results showed that CD15+ neutrophils made up 73% of ORM1+ peripheral leukocytes. In mouse lung cancer model, serum ORM1, but not liver ORM1, changed significantly in the early stage of NSCLC. ORM1 expression in peripheral leukocytes was regulated by TGF-ß and mediated by the TGF-ß/Smad signalling pathway. Our results indicated that combined ORM and TGF-ß could be a promising clinical biomarker in the diagnosis of early NSCLC.

Critical role of IL-1ß in the pathogenesis of Agrocybe aegerita galectin-induced liver injury through recruiting T cell to liver.

Acute liver failure (ALF) can be the consequence of various etiologies, which immune response plays a pivotal role in the pathogenesis. For the diversity of etiologies, more animal models are still needed in this field. Here, we developed a new acute liver injury mouse model induced by a fungal lectin AAGL (Agrocybe aegerita galectin). Intravenous injection of AAGL could induce the infiltration and activation of T, NKT and NK cells in liver and T cell played an important role in the pathogenesis. However, compared with the widely used concanavalin A model, AAGL model showed different immune mechanism. Transcriptome analysis of live tissue suggested that inflammation mediated by chemokine and cytokine signaling pathway was different between AAGL and Con A model. Fluorescent quantitative PCR verification assay showed that IL-1ß was expressed much higher in AAGL-treated mice and anti-IL-1ß could ameliorate AAGL-induced liver injury by inhibiting NF-κB and p38 signaling pathway. The expression of CXCL9 which was responsible for T cell infiltration in liver was also inhibited in AAGL model. We found a critical role of IL-1ß in the pathogenesis of AAGL model through recruiting T cells to liver, which highlighted that IL-1ß antibody might be a candidate therapy for ALF.

CBS homogenization mutation strategy narrows the glycan binding profile of a GlcNAc-specific lectin AANL.

Glycosylation plays important roles in many cellular processes, such as signal transduction, cell cycle progression and transcriptional regulation. However, the identification and analysis of glycosylation are severely hampered by the low specificity or avidity of antiglycan antibodies and lectins. We have reported that a lectin AANL, which has high specificity for terminal GlcNAc glycans and contains six carbohydrate binding sites (CBSs), was used to enrich O-GlcNAcylated peptides. To further improve AANL binding specificity, we designed a CBS-homogenization strategy and restructured six mutant lectins, known as AANL1-AANL6. Affinity chromatography with GlcNAc and isothermal titration calorimetry analysis indicated that the two mutants (AANL3 and AANL6) all maintained GlcNAc binding activity. AANL6 and AANL3 showed higher specificity for terminal GlcNAc glycans than AANL, as shown by the hemagglutination assay, cell binding assays and glycan microarray analysis, and AANL6 exhibited the highest specificity. The binding activity of AANL6 for O-GlcNAcylated peptides was shown by surface plasmon resonance assays. By AANL6 affinity chromatography enrichment and mass spectrometry analysis, 79 high-confidence and 21 putative O-GlcNAcylated sites were identified on 85 peptides mapped onto 54 proteins. Most of these sites were new sites compared with reported data. These results indicate that the enrichment capacity of AANL6 is higher than that of wild-type AANL. In conclusion, the CBS-homogenization mutation strategy was successful, and AANL6 was identified as a powerful tool for O-GlcNAcylation enrichment. Our research suggests that the CBS-homogenization strategy is valuable for improving the specificity of lectins with multiple CBSs.

Novel reversibly switchable wettability of superhydrophobic-superhydrophilic surfaces induced by charge injection and heating.

Reversibly switching wettability between superhydrophobicity and superhydrophilicity has attracted widespread interest because of its important applications. In this work, we propose a reversible superhydrophobic-superhydrophilic conversion induced by charge injection and heating. Different from the conventional electrowetting phenomenon caused by the accumulation of solid-liquid interfacial charges, we discovered a phenomenon where charge injection and accumulation at the solid surface results in a sharp increase in wettability. The wettability of a sprayed SiO2 nanoparticle coating on a glass slide was shown to change from superhydrophobic to superhydrophilic by charge injection and heating, and the superhydrophobicity was restored by heating, verifying a reversible superhydrophobic-superhydrophilic conversion. The influence of voltage, temperature, and time on the coating wettability and its durability under reversible conversion have been studied.

Identification a novel clinical biomarker in early diagnosis of human non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a malignant tumor with high morbidity and mortality. The clinical biomarkers currently used for the early diagnosis of lung cancer have poor sensitivity and specificity. Therefore, it is urgent to identify sensitive biomarkers for the early detection of NSCLC to improve the patient survival of patients. In our previously study, we identified glycoprotein alpha-1-antichymotrypsin (AACT) as an early biomarker of NSCLC. In this study, serum glycopeptides were enriched using the high-GlcNAc-specific binding lectin, AANL/AAL2, for further quantitative proteomics analysis using LC-MS/MS. A total of 55 differentially expressed proteins were identified by using demethylation labelling proteomics. Serum paraoxonase/arylesterase 1 (PON1) was selected for validation by western blotting and lectin-ELISA in samples from 120 enrolled patients. Our data showed that AANL-enriched PON1 has better diagnostic performance than total PON1 in early NSCLC, since it differed between early Stage I tumor samples and tumor-free samples (healthy and benign). Combining AANL-enriched PON1 with carcinoembryonic antigen (CEA) significantly improved the diagnostic specificity of CEA. Moreover, combined AANL-enriched PON1 and AANL-enriched AACT was significantly different between early NSCLC samples and tumor-free samples with an AUC of 0.940, 94.4% sensitivity, and 90.2% specificity. Our findings suggest that combined AANL-enriched PON1 and AANL-enriched AACT is a potential clinical biomarker for the early diagnosis of NSCLC.

In Situ Imaging of Photocatalytic Activity at Titanium Dioxide Nanotubes Using Scanning Ion Conductance Microscopy.

In this letter, in situ imaging of the photocatalytic activity of titanium dioxide (TiO2) nanotubes for the degradation of an organic pollutant (i.e., Rhodamine B (RhB)) is realized with nanometer resolution using scanning ion conductance microscopy (SICM). Upon illumination, the separated electrons and holes at the nanotubes induce oxidation of RhB to produce the more positively charged Rhodamine 123 (Rh 123), which leads to increased ionic current through the capillary orifice and an elevated apparent altitude in the SICM image. Active sites with higher activity on the nanotubes exhibit a significant high spatial-resolution character. The successful imaging of the photocatalytic activity of TiO2 nanotubes should provide an in situ approach for local investigation of the photocatalytic process at the catalyst.

Bldesin, the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities.

Fungus defensin is a kind of important natural peptide resource, such as plectasin from the soil fungus Pseudoplectania nigrella with potential application in the antimicrobial peptide lead drug discovery. Here, a fungus defensin named Bldesin with Kv1.3 channel and serine protease inhibitory activities was first explored. By GST-Bldesin fusion expression and enterokinase cleaving strategy, recombinant Bldesin was obtained successfully. Antimicrobial assays showed that Bldesin had potent activity against Gram-positive Staphylococcus aureus, but had no effect on Gram-negative Escherichia coli. Electrophysiological experiments showed that Bldesin had Kv1.3 channel inhibitory activity. Serine protease inhibitory associated experiments showed that Bldesin had unique chymotrypsin protease inhibitory, elastase protease inhibitory, and serine protease-associated coagulation inhibitory activities. To the best of our knowledge, Bldesin is the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities and highlighted novel pharmacological effects of fungus-derived defensin peptides.