Antifungal Peptide Research Articles

P379 Antifungal activity of α defensins 5 like peptide against Cryptococcus neoformans var. grubii and in silico designing of its peptidomimetics

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesCombinatorial and retroviral therapies have been found effective in the treatment of cryptococcosis, but safer and more potent antifungal therapeutics are the need of the hour. In this regard, the natural antifungal peptides (AFPs), the essential components of the innate immune system have drawn the attention of researchers and drug developers. AFPs are 12-50 amino-acids-long host defense peptides of cationic nature. Although AFPs have certain limitations like low stability, susceptibility to proteolysis, loss of activity in the presence of metal ions and salts, short half-lives, and peptide aggregation in oral formulation, but these can be addressed by designing and synthesizing the peptidomimetics.MethodologyThe whole blood of the rat was collected by cardiac puncture. The blood was mixed with 0.83% aqueous ammonium chloride. The neutrophils were separated from lysed WBCs by centrifugation. The purification of the peptide was carried out by SDS-PAGE, size exclusion, cation exchange chromatography, and HRLCMS. The MSA of the characterized peptide was performed using a T-coffee program. The in vitro antifungal activity of crude was tested against C. n. grubii (ATCC 6352) using flow cytometry. The structure of peptides was altered through ChemDraw by replacing the elements with their bioisosteres to form mimetics. The peptide and its mimetics were docked upon the target of C. n. grubii, i.e., O-acetyl transferase (modeled through Phyre2 web server) using PatchDock, and FireDock web servers.ResultsThe peptide isolated from rat PMNs had a molecular weight of 3 kDa. It has lysine and arginine which are the characteristic residues of an antifungal peptide. The eluted peptide has the highest (83%) similarity with α defensins 5 (accession no NP_001013071 XP_214 386). The mild antifungal activity of peptide was evident against C. n. grubii. The structure of O-acetyl transferase was modeled with 100% confidence and 92% query coverage (Table 1). The mimetic 1 (M1) showed the best interaction with O-acetyl transferase with −54.47 Kcal/mol binding energy and 4 hydrogen bonds (Table 2).ConclusionThe designed M1 of α defensins 5 like peptide could be a promising lead in antifungal drug development. As mimetic 1 was designed using an in-silico approach, its synthesis and wet lab validation are desirable.

P003 Synthetic antifungal peptide mimic kills Candida albicans by targeting protein glycosylation and synergistically prevents infection

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMFungal infections represent a serious burden on human health. Increasing numbers of susceptible hosts, a limited set of approved antifungal drugs which frequently trigger undesired side effects, and the emergence of resistant strains highlight the urgent demand for novel antifungal drug formulations. However, the biological similarity of human and fungal cells hampers the development of new antifungals which do not also harm humans. In nature, organisms in almost all domains of life produce antimicrobial peptides to combat microbial pathogens. Those peptides share certain characteristics, such as being short, amphiphilic molecules with a positive net charge.1 We designed synthetic polyacrylamides which mimic the properties of naturally occurring antifungal peptides. These positively charged, amphiphilic polymers are advantageous over peptides because of their easy synthesis and stability against proteases. Initial structure-activity relationship studies revealed an optimal cLogP (the calculated hydrophobicity of a molecule) around 1.5 to ensure activity against C. albicans and simultaneous biocompatibility with host cells.2 Additionally, shorter polymers with a length of 20 subunits were more effective than their longer versions.2 In terms of their therapeutic index, certain compositions outperformed the broad-spectrum antifungal amphotericin B and were even effective against drug-resistant clinical isolates of C. albicans.2 Candida albicans strains with known antifungal drug-resistance mutations were not affected in their susceptibility to the polymers. Therefore, investigations were carried out to elucidate the mode of action of the polymers. The transcriptome of C. albicans cells treated with subinhibitory concentrations of the polymers revealed an increased expression of genes involved in general stress response and upregulation in protein processing in the endoplasmic reticulum, particularly glycosylation and degradation. These findings, together with electron microscopy observations, indicated damage to the mannoproteins in the cell wall of the fungus. Membrane damage was also observed utilizing a C. albicans strain expressing GFP intracellularly. The in vitro therapeutic potential of the most promising polymer was tested in a human epithelial cell (HEC) model simulating C. albicans infection. The polymer alone was not able to prevent C. albicans infection of HECs. However, the combination of polymer with caspofungin or fluconazole showed very strong synergistic effects at otherwise non-inhibitory concentrations of the individual antifungals, successfully stopping fungal infection in vitro without damaging the HECs. These results underline the potential of synthetic polymers as an alternative treatment for fungal infections with low toxicity to human cells and a novel mode of action.Sources1. Fernández de Ullivarri, M., Arbulu, S., Garcia-Gutierrez, E. and Cotter, P.D. Antifungal peptides as therapeutic agents. Front Cell Infect Microbiol 10, 00 105 (2020).2. Schaefer, S. et al. Rational design of an antifungal polyacrylamide library with reduced host-cell toxicity. ACS Appl Mater Interfaces 13, 27430-27444 (2021).

RNAi of Mannosidase-Ia in the Colorado potato beetle and changes in the midgut and peritrophic membrane.

In addition to its role in the digestive system, the peritrophic membrane (PM) provides a physical barrier protecting the intestine from abrasion and against pathogens. Because of its sensitivity to RNA interference (RNAi), the notorious pest insect, the Colorado potato beetle (CPB, Leptinotarsa decemlineata), has become a model insect for functional studies. Previously, RNAi-mediated silencing of Mannosidase-Ia (ManIa), a key enzyme in the transition from high-mannose glycan moieties to paucimannose N-glycans, was shown to disrupt the transition from larva to pupa and the metamorphosis into adult beetles. While these effects at the organismal level were interesting in a pest control context, the effects at the organ or tissue level and also immune effects have not been investigated yet. To fill this knowledge gap, we performed an analysis of the midgut and PM in ManIa-silenced insects. As marked phenotype, the ManIaRNAi insects, the PM pore size was found to be decreased when compared to the control GFPRNAi insects. These smaller pores are related to the observation of thinner microvilli (Mv) on the epithelial cells of the midgut of ManIaRNAi insects. A midgut and PM proteome study and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis with a selection of marker genes was performed to characterize the midgut cells and understand their response to the silencing of ManIa. In agreement with the loss of ManIa activity, an accumulation of high-mannose N-glycans was observed in the ManIa-silenced insects. As a pathogen-associated molecular pattern (PAMP), the presence of these glycan structures could trigger the activation of the immune pathways. The observed decrease in PM pore size could be a response to prevent potential pathogens to access the midgut epithelium. This hypothesis is supported by the strong increase in transcription levels of the anti-fungal peptide drosomycin-like in ManIaRNAi insects, although further research is required to elucidate this possibility. The potential immune response in the midgut and the smaller pore size in the PM shed a light on the function of the PM as a physical barrier and provide evidence for the relation between the Mv and PM. © 2022 Society of Chemical Industry.

Antifungal Peptide P852 Controls Fusarium Wilt in Faba Bean (Viciafaba L.) by Promoting Antioxidant Defense and Isoquinoline Alkaloid, Betaine, and Arginine Biosyntheses.

Green pesticides are highly desirable, as they are environmentally friendly and efficient. In this study, the antifungal peptide P852 was employed to suppress Fusarium wilt in the Faba bean. The disease index and a range of physiological and metabolomic analyses were performed to explore the interactions between P852 and the fungal disease. The incidence and disease index of Fusarium wilt were substantially decreased in diseased Faba beans that were treated with two different concentrations of P852 in both the climate chamber and field trial. For the first time, P852 exhibited potent antifungal effects on Fusarium in an open field condition. To explore the mechanisms that underlie P852′s antifungal effects, P852 treatment was found to significantly enhance antioxidant enzyme capacities including guaiacol peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and the activities of antifungal enzymes including chitinase and β-1,3-glucanase, as well as plant dry and fresh weights, and chlorophyll content compared to the control group (p ≤ 0.05). Metabolomics analysis of the diseased Faba bean treated with P852 showed changes in the TCA cycle, biological pathways, and many primary and secondary metabolites. The Faba bean treated with a low concentration of P852 (1 μg/mL, IC50) led to upregulated arginine and isoquinoline alkaloid biosynthesis, whereas those treated with a high concentration of P852 (10 μg/mL, MFC) exhibited enhanced betaine and arginine accumulation. Taken together, these findings suggest that P852 induces plant tolerance under Fusarium attack by enhancing the activities of antioxidant and antifungal enzymes, and restoring plant growth and development.

32994 Pezadeftide is a potent antifungal peptide with rapid fungicidial activity via a unique mechanism of action

Background: Fungal infections of skin, hair, and nails are extremely common and can be caused by a broad range of fungal pathogens including dermatophytes, yeasts, and nondermatophytic molds. Pezadeftide is a plant defensin peptide with potent fungicidal activity against a range of fungal pathogens that cause skin and nail infections, including Trichophyton spp., Candida spp, and a range of nondermatophytic molds. Pezadeftide is currently in development as a new topical treatment for onychomycosis. Methods: The activity of pezadeftide against a range of fungal pathogens was tested using the microbroth dilution method to determine the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). Fluorescent dyes were employed to assess the effect of pezadeftide on various cellular components. To identify potential targets for pezadeftide, a yeast deletion library was screened for fitness in the presence of low concentrations of pezadeftide. Results: Pezadeftide has potent antifungal activity across a broad range of fungal pathogens that cause dermatophytic infections. Pezadeftide inhibits growth at low micromolar concentrations and is fungicidal at similar concentrations, suggesting that its primary mode-of-action is fungicidal. Pezadeftide rapidly enters fungal cells and causes a rapid mitochondrial response that results in hyperpolarization of the mitochondrial membrane. Fusion of the vacuoles and production of reactive oxygen species are observed prior to disruption of the plasma membrane and cell death. Conclusions: Pezadeftide is a potent, broad-spectrum antifungal with a fungicidal mode-of-action. It rapidly kills fungal cells via interaction with the mitochondria, hyperpolarisation of the mitochondrial membrane and production of reactive oxygen species.

Identification of Pseudo-R genes in Vitis vinifera and characterization of their role as immunomodulators in host-pathogen interactions

IntroductionDuplication events are fundamental to co-evolution in host-pathogen interactions. Pseudogenes (Ψs) are dysfunctional paralogs of functional genes and resistance genes (Rs) in plants are the key to disarming pathogenic invasions. Thus, deciphering the roles of pseudo-R genes in plant defense is momentous. ObjectivesThis study aimed to functionally characterize diverse roles of the resistance Ψs as novel gene footprints and as significant gene regulators in the grapevine genome. MethodsPlantPseudo pipeline and HMM-profiling identified whole-genome duplication-derived (WGD) Ψs associated with resistance genes (Ψ-Rs). Further, novel antifungal and antimicrobial peptides were characterized for fungal associations using protein–protein docking with Erysiphe necator proteins. miRNA and tasiRNA target sites and transcription factor (TF) binding sites were predicted in Ψ-Rs. Finally, differential co-expression patterns in Ψ-Rs-lncRNAs-coding genes were identified using the UPGMA method. Results2,746 Ψ-Rs were identified from 31,032 WGD Ψs in the genome of grapevine. 69-antimicrobial and 81-antifungal novel peptides were generated from Ψ-Rs. The putative genic potential was predicted for five novel antifungal peptides which were further characterized by docking against E. necator proteins. 395 out of 527 resistance loci-specific Ψ-Rs were acting as parental gene mimics. Further, to explore the diverse roles of Ψ-Rs in plant-defense, we identified 37,026 TF-binding sites, 208 miRNA, and 99 tasiRNA targeting sites on these Ψ-Rs. 194 Ψ-Rs were exhibiting tissue-specific expression patterns. The co-expression network analysis between Ψs-lncRNA-genes revealed six out of 79 pathogen-responsive Ψ-Rs as significant during pathogen invasion. ConclusionsOur study provides pathogen responsive Ψ-Rs integral for pathogen invasion, which will offer a useful resource for future experimental validations. In addition, our findings on novel peptide generations from Ψ-Rs offer valuable insights which can serve as a useful resource for predicting novel genes with the futuristic potential of being investigated for their bioactivities in the plant system.

Structural insights into the interaction of antifungal peptides and ergosterol containing fungal membrane

The treatment of invasive drug-resistant and potentially life-threatening fungal infections is limited to few therapeutic options that are usually associated with severe side effects. The development of new effective antimycotics with a more tolerable side effect profile is therefore of utmost clinical importance. Here, we used a combination of complementary in vitro assays and structural analytical methods to analyze the interaction of the de novo antimicrobial peptide VG16KRKP with the sterol moieties of biological cell membranes. We demonstrate that VG16KRKP disturbs the structural integrity of fungal membranes both invitro and in model membrane system containing ergosterol along with phosphatidylethanolamine lipid and exhibits broad-spectrum antifungal activity. As revealed by systematic structure-function analysis of mutated VG16KRKP analogs, a specific pattern of basic and hydrophobic amino acid side chains in the primary peptide sequence determines the selectivity of VG16KRKP for fungal specific membranes.

A β-defensin isoform from the Flathead grey mullet, Mugil cephalus (Linnaeus 1758): Structural and functional characterisation in silico

Defensins are an important class of AMPs, rich in cysteine with antibacterial, antifungal and antiviral functions. The present study identified and functionally characterised a β-defensin gene from Grey mullet, Mugil cephalus, termed as Mc-Def. A 192 base pair complementary DNA with an open reading frame encoding 63 amino acids comprising 20 amino acid signal and 43 amino acid mature peptide region was obtained. Mc-Def has a net charge of 3.9 and a molecular weight of 7.27 kDa with an isoelectric point of 8.88. It possessed six cysteines forming 3 disulphide bonds between C1–C5, C2–C4 and C3–C6. Three dimensional structure identified a short N-terminal α helix followed by β sheet strands stabilized by disulfide bonds. The mRNA structure was predicted by Biomodel server with significant MEF score. Multiple sequence alignment and phylogeny revealed the sequence similarity of Mc-Def with fish β-defensin 2 type of AMPs. Molecular diversity and evolution of β-defensin provided immunity to host system against various infections. In silico analysis identified its potential as antibacterial, antifungal, anticancer, antioxidant and non-hemolytic peptide activity. These intriguing results firmly suggest that the Mc-Def can be used as a potent drug molecule in fish health management.

Discovery and Characterization of a Potent Antifungal Peptide through One-Bead, One-Compound Combinatorial Library Screening.

This work describes the discovery of a bead-bound membrane-active peptide (MAP), LBF127, that selectively binds fungal giant unilamellar vesicles (GUVs) over mammalian GUVs. LBF127 was re-synthesized in solution form and demonstrated to have antifungal activity with limited hemolytic activity and cytotoxicity against mammalian cells. Through systematic structure-activity relationship studies, including N- and C-terminal truncation, alanine-walk, and d-amino acid substitution, an optimized peptide, K-oLBF127, with higher potency, less hemolytic activity, and cytotoxicity emerged. Compared to the parent peptide, K-oLBF127 is shorter by three amino acids and has a lysine at the N-terminus to confer an additional positive charge. K-oLBF127 was found to have improved selectivity toward the fungal membrane over mammalian membranes by 2-fold compared to LBF127. Further characterizations revealed that, while K-oLBF127 exhibits a spectrum of antifungal activity similar to that of the original peptide, it has lower hemolytic activity and cytotoxicity against mammalian cells. Mice infected with Cryptococcus neoformans and treated with K-oLBF127 (16 mg/kg) for 48 h had significantly lower lung fungal burden compared to untreated animals, consistent with K-oLBF127 being active in vivo. Our study demonstrates the success of the one-bead, one-compound high-throughput strategy and sequential screening at identifying MAPs with strong antifungal activities.

Identification of a New Antifungal Peptide W1 From a Marine Bacillus amyloliquefaciens Reveals Its Potential in Controlling Fungal Plant Diseases.

A bacterium, Bacillus amyloliquefaciens W0101, isolated from the Arctic Ocean, showed potent antifungal activity against several plant pathogenic fungi. An antifungal peptide W1, with a molecular weight of approximately 2.4 kDa, was purified from the culture supernatant of the strain W0101 using ion-exchange chromatography and high-performance liquid chromatography. By analysis of Liquid Chromatograph-Mass Spectrometer, the peptide W1 was identified as a new antifungal peptide derived from the fragment of preprotein translocase subunit YajC. Further analysis revealed that W1 could disrupt the hyphae and spores of Sclerotinia sclerotiorum and inhibit its growth. W1 suppressed S. sclerotiorum and Fusarium oxysporum at a minimum inhibitory concentration of 140 and 58 μg/ml, respectively. The antifungal activity of W1 remained stable at 20–80°C or pH 6–11, with reduced activity at 100–110°C and pH 4–5, and under three protease treatments. Additionally, W1 also had a certain extent of metal ion resistance. These results therefore suggest that the peptide W1 from marine B. amyloliquefaciens W0101 may represent a new antifungal peptide with potential application in the biocontrol of plant diseases.

Production and fermentation characteristics of antifungal peptides by synergistic interactions with Lactobacillus paracasei and Propionibacterium freudenii in supplemented whey protein formulations

Whey protein, as a nutrient-rich waste from dairy industry, several studies have investigated its bioconversion. This study aimed to convert whey protein into antifungal peptides by synergistic interactions with Lactobacillus paracasei and Propionibacterium freudenreichii. The response surface methodology was employed for optimization. The optimum fermentation medium contained 26.69 g/L lactose, 28.44 g/L nitrogen sources (soytone: whey protein = 2:1, m/m) and the inoculum size was at 3.72% (v/v). The fermentate was sensitive to proteolytic enzymes, strong alkaline (pH > 10.0) and high temperature. A total of 156 peptides were identified by nanoliter liquid phase-Q Exactive quadrupole ultra-high resolution orbitrap mass spectrometer (Nano LC-Q Exactive Plus) and screened by PEAKS X software. Finally, the decisive factors of antifungal function were determined by principal component analysis. The research provided experimental and theoretical support for the process optimization and mechanistic explanation of whey protein fermentation to produce antifungal substances.

Genome-wide analysis of PTR transporters in Candida species and their functional characterization in Candida auris.

The peptide transport (PTR) or proton-dependent oligopeptide transporter (POT) family exploits the inwardly directed proton motive force to facilitate the cellular uptake of di/tripeptides. Interestingly, some representatives are also shown to import peptide-based antifungals in certain Candida species. Thus, the identification and characterization of PTR transporters serve as an essential first step for their potential usage as antifungal peptide uptake systems. Herein, we present a genome-wide inventory of the PTR transporters in five prominent Candida species. Our study identifies 2 PTR transporters each in C. albicans and C. dubliniensis, 1 in C. glabrata, 4 in C. parapsilosis, and 3 in C. auris. Notably, despite all representatives retaining the conserved features seen in the PTR family, there exist two distinct classes of PTR transporters that differ in terms of their sequence identities and lengths of certain extracellular and intracellular segments. Further, we also evaluated the contribution of each PTR protein of the newly emerged multi-drug-resistant C. auris in di/tripeptide uptake. Notably, deletion of two PTR genes BNJ08_003830 and BNJ08_005124 led to a marked reduction in the transport capabilities of several tested di/tripeptides. However, all three genes could complement the role of native PTR2 gene of Saccharomyces cerevisiae, albeit to varied levels. Besides, BNJ08_005124 deletion also resulted in increased resistance toward the peptide-nucleoside drug Nikkomycin Z as well as the glucosamine-6-phosphate synthase inhibitor, L-norvalyl-N3-(4-methoxyfumaroyl)-L-2,3-diaminopropionoic acid (Nva-FMDP), pointing toward its predominant role in their uptake mechanism. Altogether, the study provides an important template for future structure-function investigations of PTR transporters in Candida species. KEY POINTS: • Candida genome encodes for two distinct classes of PTR transporters. • Candida auris encodes for 3 PTR transporters with different specificities. • BNJ08_005124 in C. auris is involved in the uptake of Nikkomycin Z and Nva-FMDP.

Role of Vaginal Mucosa, Host Immunity and Microbiota in Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) is a prevalent gynaecological disease characterised by vaginal wall inflammation that is caused by Candida species. VVC impacts almost three-quarters of all women throughout their reproductive years. As the vaginal mucosa is the first point of contact with microbes, vaginal epithelial cells are the first line of defence against opportunistic Candida infection by providing a physical barrier and mounting immunological responses. The mechanisms of defence against this infection are displayed through the rapid shedding of epithelial cells, the presence of pattern recognition receptors, and the release of inflammatory cytokines. The bacterial microbiota within the mucosal layer presents another form of defence mechanism within the vagina through acidic pH regulation, the release of antifungal peptides and physiological control against dysbiosis. The significant role of the microbiota in maintaining vaginal health promotes its application as one of the potential treatment modalities against VVC with the hope of alleviating the burden of VVC, especially the recurrent disease. This review discusses and summarises current progress in understanding the role of vaginal mucosa and host immunity upon infection, together with the function of vaginal microbiota in VVC.

Antifungal Activity of NP20 Derived from Amphioxus Midkine/Pleiotrophin Homolog Against Aspergillus niger and Aspergillus fumigatus.

With the emergence of antifungal resistance, systematic infections with Aspergillus are becoming the major cause of the clinical morbidity. The development of novel antifungal agents with high efficacy, low drug tolerance, and few side effects is urgent. In response to that need, we have identified NP20. Here we demonstrate clearly that NP20 has antifungal activity, capable of killing the spores of Aspergillus niger and Aspergillus fumigatus as well as causing direct damage to the surface, membrane, cytoplasm, organelle, and nucleus of the fungal spores. Interestingly, NP20 is active under temperature stress and a wide range of pH. Subsequently, MTT assay, assay for binding of NP20 to fungal cell wall components, membrane depolarization assay, confocal microscopy, ROS assay, DNA replication, and protein synthesis assay are performed to clarify the mechanisms underlying NP20 against Aspergillus. The results show that NP20 can bind with and pass through the fungal cell wall, and then interfere with the lipid membrane. Moreover, NP20 can induce intracellular ROS production, DNA fragmentation, and protein synthesis inhibition of the fungal cells. These together indicate that NP20 is a novel antifungal peptide, which has considerable potential for future development as novel peptide antibiotics against Aspergillus.

Antibacterial and antifungal activity of kenaf seed peptides and their effect on microbiological safety and physicochemical properties of some food models

This study aimed to develop natural antibacterial and antifungal peptides from kenaf seed powder and to determine their efficacy in prolonging the shelf-life of fruit juices and bread systems. The kenaf seed peptides mixture (KSPM) displayed potent antimicrobial activity ranging from 94.48 to 98.99% against Gram-positive and Gram-negative bacteria and fungi. Sixteen low molecular weight cationic peptides ranging from 768 to 1416 Da were identified, in which nine of them were de novo peptides. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of KSPM against gram-positive bacteria were 11 μg/mL, 22 μg/mL for gram-negative bacteria, with a higher MIC of 43 μg/mL and minimum fungicidal concentration (MFC) of 86 μg/mL required against fungi. The KSPM displayed a rapid bactericidal effect against all the tested bacteria at 1 MIC within 1 h, and a rapid fungicidal effect against all the tested fungi at 2 MIC within 4 h. The KSPM retained a 100% of its activity up to 121 °C, 100 mM NaCl and in an acidic environment, and to a lesser extent of 64–72% under neutral condition. The peptides were fully digested by gastrointestinal proteases including pepsin, trypsin and α-chymotrypsin. The incorporation of 1000 and 3000 mg/kg KSPM in bacteria-inoculated mango and pineapple juices caused a ≥5-log10 reduction in the viable counts of Salmonella typhimurium, Escherichia coli and Listeria monocytogenes within 1–2 h during storage at 4 °C. The application of KSPM at 3000 mg/kg reduced Aspergillus niger, Aspergillus flavus and Fusarium sp. count by 3–4 log10 in fungi-inoculated bread samples when stored at 25 °C. The peptides at 1000 and 3000 mg/kg successfully extended the shelf-life of bacteria-free fruit juices for 150 days, and mould-free bread for 10 and 16 days. The addition of KSPM to fruit juices and bread model did not induce alteration to their physicochemical properties. These findings demonstrating the potential application of KSPM as a natural preservative in the food industry.

Production of Demineralized Antibacterial, Antifungal and Antioxidant Peptides from Bovine Hemoglobin Using an Optimized Multiple-Step System: Electrodialysis with Bipolar Membrane.

Numerous studies have shown that bovine hemoglobin, a protein from slaughterhouse waste, has important biological potential after conventional enzymatic hydrolysis. However, the active peptides could not be considered pure since they contained mineral salts. Therefore, an optimized multi-step process of electrodialysis with bipolar membranes (EDBM) was carried out to produce discolored and demineralized peptides without the addition of chemical agents. The aim of this study was to test the antibacterial, antifungal and antioxidant activities of discolored and demineralized bovine hemoglobin hydrolysates recovered by EDBM and to compare them with raw and discolored hydrolysates derived from conventional hydrolysis. The results demonstrate that discolored–demineralized hydrolysates recovered from EDBM had significant antimicrobial activity against many bacterial (gram-positive and gram-negative) and fungal (molds and yeast) strains. Concerning antibacterial activity, lower MIC values for hydrolysates were registered against Staphylococcus aureus, Kocuria rhizophila and Listeria monocytogenes. For antifungal activity, lower MIC values for hydrolysates were registered against Paecilomyces spp., Rhodotorula mucilaginosa and Mucor racemosus. Hemoglobin hydrolysates showed fungicidal mechanisms towards these fungal strains since the MFC/MIC ratio was ≤4. The hydrolysates also showed a potent antioxidant effect in four different antioxidant tests. Consequently, they can be considered promising natural, low-salt food preservatives. To the best of our knowledge, no previous studies have identified the biological properties of discolored and demineralized bovine hemoglobin hydrolysates.

Peptide-based edible coatings to control postharvest fungal spoilage of mango (Mangifera indica L.) fruit

Edible coatings have been applied to improve the shelf life and quality of fruit. However, little is known about the potential of bioactive peptides-based coatings for the fruit preservation. This study aimed to evaluate the antifungal activity of edible coatings produced by incorporating bioactive peptides generated by lacto-fermentation of palm kernel cake (PKC) fermented with Lactobacillus plantarum ATCC8014 (PKCL1) and Lactobacillus fermentum ATCC9338 (PKCL2). PKCL1 and PKCL2 incorporated with 10 different polysaccharide polymers, and evaluated the antifungal activity, biodegradability and peptide releasing ability. The results showed that the edible coating produced by incorporating PKCL1 and PKCL2 in chitosan (CH) inhibited the growth of fungi that commonly infest mangoes. CH coating containing PKCL1 and PKCL2 showed prominent inhibition zones against Colletotrichum gloeosporioides and Botryodiplodia theobromae . The incorporation of PKCL1 and PKCL2 CH matrix significantly ( p ˂ 0.05 ) reduced anthracnose and stem-end rot. Furthermore, the peptide release and biodegradability of CH-based coating were significantly higher than coatings produced using 9 other polysaccharides. The incorporation of PKCL1 and PKCL2 into CH significantly altered the color and water vapor permeability polysaccharide-based films, although the swelling property was only changed marginally. Out of 10 polysaccharides testes, CH was found to be the best carrier for antifungal peptides. CH coatings containing PKCL1 and PKCL2 demonstrated a high potential to control the postharvest anthracnose and stem-end rot in mango. However, further research should be carried out, focusing on the quality and sensory properties of treated mango. • Edible coatings incorporating lacto-fermented peptides demonstrated antifungal activity. • The antifungal edible coating inhibited the growth of C. gloeosporioides and B. theobromae . • The antifungal edible coating significantly ( P ˂ 0.05) reduced anthracnose and stem-end rot in mango. • Chitosan was the best polysaccharide carrier for the antifungal peptides.

The potential of antifungal peptide Sesquin as natural food preservative

Sesquin is a wide spectrum antimicrobial peptide displaying a remarkable activity on fungi. Contrarily to most antimicrobial peptides, it presents an overall negative charge. In the present study, we elucidate the molecular basis of its mode of action towards biomimetic membranes by NMR and MD experiments. While a specific recognition of phosphatidylethanolamine (PE) might explain its activity in a variety of different organisms (including bacteria), a further interaction with ergosterol accounts for its strong antifungal activity. NMR data reveal a charge gradient along its amide protons allowing the peptide to reach the membrane phosphate groups despite its negative charge. Subsequently, the peptide gets structured inside the bilayer, reducing its order. MD simulations predict that its activity is retained in conditions commonly used for food preservation: low temperatures, high pressure, or the presence of electric field pulses, making Sesquin a good candidate as food preservative.

Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development.

ABSTRACTCryptococcus neoformans is a major fungal pathogen that often causes life-threatening meningitis in immunocompromised populations. This yeast pathogen is highly resistant to the echinocandin drug caspofungin. Previous studies showed that Cryptococcus lipid translocase (flippase) is required for the caspofungin resistance of that fungus. Mutants with a deleted subunit of lipid flippase, Cdc50, showed increased sensitivity to caspofungin. Here we designed an antifungal peptide targeting the P4-ATPase function. We synthesized stable peptides based on the Cdc50 loop region to identify peptides that can sensitize caspofungin by blocking flippase function and found that myristylated peptides based on the “AS15 sequence” was effective at high concentrations. A modified peptide, “AW9-Ma” showed a MIC of 64 μg/mL against H99 wild type and a fractional inhibitory concentration (FIC) index value of 0.5 when used in combination with caspofungin. Most notably, in the presence of the AW9-Ma peptide, C. neoformans wild type was highly sensitive to caspofungin with a MIC of 4 μg/mL, the same as the cdc50Δ mutant. Further assays with flow cytometry showed inhibition of the lipid flippase enzyme activity and significant accumulation of phosphatidylserine on the cell membrane surface. Using a fluorescently labeled peptide, we confirmed that the peptide co-localized with mCherry-tagged P4-ATPase protein Apt1 in C. neoformans. Structure-activity relationship studies of the AW9 sequence showed that two lysine residues on the peptide are likely responsible for the interaction with the P4-ATPase, hence critical for its antifungal activity.IMPORTANCE The authors have developed a lead compound peptide antifungal drug targeting a protein from the organism Cryptococcus neoformans. Binding of the drug to the target fungal protein causes charged lipid molecules to be retained on the surface. This peptide works in synergy with the existing antifungal drug caspofungin. Echinocandin drugs like caspofungin are one of the few classes of existing antifungals. Due to the high concentrations needed, caspofungin is rarely used to treat C. neoformans infections. The authors believe that their new compound provides a way to lower the concentration of caspofungin needed to treat such infections, thus opening the possibility for greater utility of these antifungal.

Analysis of the effects of antifungal peptide P-1 from Bacillus pumilus HN-10 on energy metabolism of Trichothecium roseum

This study investigated the antifungal activity and possible mode of action of Bacillus pumilus HN-10 antifungal peptide P-1 against Trichothecium roseum. The results showed that the antifungal peptide P-1 at a concentration of 1.0 μg mL-1 had strong antifungal activity against T. roseum. P-1 inhibited the tricarboxylic acid cycle (TCA) pathway and the transporter pathway of NADH to coenzyme Q on the electron transport chain. P-1 significantly reduced succinate dehydrogenase (SDH), malate dehydrogenase (MDH), ATPase, mitochondrial complex enzymes I, II and IV enzyme activities on the electron transport chain, and 5'-triphosphate (ATP), 5'-diphosphate (ADP), 5'-monophosphate (AMP) content, and energy charge (EC); significantly increased 6-phosphofructokinase (PFK) enzyme activity. The release of Ca2+ (OD680) from the inner mitochondrial membrane and the openness of the mitochondrial permeability transition pore (MPTP) were analysed, and microscopy was performed following staining of mitochondria with JC-1. The results indicated that P-1 significantly increased the release of Ca2+ and the openness of MPTP, decreased the mitochondrial membrane potential, and produced green fluorescence; transcriptomics data analysis showed that there were 39 differentially expressed genes (DEGs) related to energy metabolism enzymes. The results verified by qRT-PCR were basically consistent with the transcriptome sequencing results. Thus, P-1 achieved its inhibitory effect mainly by regulating genes related to energy metabolism.