Defensin Peptides Research Articles

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.

Keratinocyte-derived defensins activate neutrophil-specific receptors Mrgpra2a/b to prevent skin dysbiosis and bacterial infection

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S.aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1β and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Membrane Permeabilization and Antimicrobial Activity of Recombinant Defensin-d2 and Actifensin against Multidrug-Resistant Pseudomonas aeruginosa and Candida albicans.

Antimicrobial resistance requires urgent efforts towards the discovery of active antimicrobials, and the development of strategies to sustainably produce them. Defensin and defensin-like antimicrobial peptides (AMPs) are increasingly gaining pharmacological interest because of their potency against pathogens. In this study, we expressed two AMPs: defensin-d2 derived from spinach, and defensin-like actifensin from Actinomyces ruminicola. Recombinant pTXB1 plasmids carrying the target genes encoding defensin-d2 and actifensin were generated by the MEGAWHOP cloning strategy. Each AMP was first expressed as a fusion protein in Escherichia coli, purified by affinity chromatography, and was thereafter assayed for antimicrobial activity against multidrug-resistant (MDR) pathogens. Approximately 985 µg/mL and 2895 µg/mL of recombinant defensin-d2 and actifensin, respectively, were recovered with high purity. An analysis by MALDI-TOF MS showed distinct peaks corresponding to molecular weights of approximately 4.1 kDa for actifensin and 5.8 kDa for defensin-d2. An in vitro antimicrobial assay showed that MDR Pseudomonas aeruginosa and Candida albicans were inhibited at minimum concentrations of 7.5 µg/mL and 23 µg/mL for recombinant defensin-d2 and actifensin, respectively. The inhibitory kinetics of the peptides revealed cidal activity within 4 h of the contact time. Furthermore, both peptides exhibited an antagonistic interaction, which could be attributed to their affinities for similar ligands, as deduced by peptide–ligand profiling. Moreover, both peptides inhibited biofilm formation, and they exhibited no resistance potential and low hemolytic activity. The peptides also possess the ability to permeate and disrupt the cell membranes of MDR P. aeruginosa and C. albicans. Therefore, recombinant actifensin and defensin-d2 exhibit broad-spectrum antimicrobial activity and have the potential to be used as therapy against MDR pathogens.

Recent development of machine learning-based methods for the prediction of defensin family and subfamily.

Nearly all living species comprise of host defense peptides called defensins, that are crucial for innate immunity. These peptides work by activating the immune system which kills the microbes directly or indirectly, thus providing protection to the host. Thus far, numerous preclinical and clinical trials for peptide-based drugs are currently being evaluated. Although, experimental methods can help to precisely identify the defensin peptide family and subfamily, these approaches are often time-consuming and cost-ineffective. On the other hand, machine learning (ML) methods are able to effectively employ protein sequence information without the knowledge of a protein's three-dimensional structure, thus highlighting their predictive ability for the large-scale identification. To date, several ML methods have been developed for the in silico identification of the defensin peptide family and subfamily. Therefore, summarizing the advantages and disadvantages of the existing methods is urgently needed in order to provide useful suggestions for the development and improvement of new computational models for the identification of the defensin peptide family and subfamily. With this goal in mind, we first provide a comprehensive survey on a collection of six state-of-the-art computational approaches for predicting the defensin peptide family and subfamily. Herein, we cover different important aspects, including the dataset quality, feature encoding methods, feature selection schemes, ML algorithms, cross-validation methods and web server availability/usability. Moreover, we provide our thoughts on the limitations of existing methods and future perspectives for improving the prediction performance and model interpretability. The insights and suggestions gained from this review are anticipated to serve as a valuable guidance for researchers for the development of more robust and useful predictors.

Necessity for modern methods of diagnosing Hepatitis C, especially when overlapping with secondary infection

In this study, we focused on the antimicrobial peptides and T-lymphocyte clusters indicating the immune system ability to react violently leading to reduce the organism sensitivity to bacteria tested in 87 individuals with chronic hepatitis C. All patients were divided into two groups: with chronic hepatitis C, and the group in which hepatitis C overlaps with secondary infection leading to pneumonia. Endotoxin and lipopolysaccharide-binding protein (LBP) were determined using the ELISA technique. Determination of the clusters of differentiation (CD) carried out by indirect immunofluorescence reaction, while the circulating immune clusters (CIC) identified by method of sedimentation with a 3.5% solution of polyethylene glycol. Statistical processing of the results carried out using the Wilkinson U-test (Mann - Whitney). In the group of patients with hepatitis C, CD25+ was nearly halved, while in group with hepatitis aggravated by pneumonia this value lowered approximately three times. CD25+ indicator in II group was even 1.4 times less than in the group without pneumonia. Defensin levels were significantly higher in the I group, where endotoxin raises up to 24.4 vs normal levels. In the II group, aggravated by pneumonia, endotoxin elevated even up to 57.7 IU/ml, the same direction changed defensin concentration. The results of this study show, that instead of standard tests for liver damage: bilirubin, AST and γ-glutamyl transpeptidase, it is much more expedient to use the antimicrobial peptides defensin and LBP, which are more informative in the diagnosis of hepatitis C, especially when it overlaps with secondary infection.

The probiotic potential of Lactobacillus plantarum strain RW1 isolated from canine faeces.

To evaluation the probiotic potential of Lactobacillus plantarum strain RW1 isolated from healthy dogs for its further utilization as a dietary supplement for dogs. This study aimed to evaluate the probiotic potential of L. plantarum strain RW1 isolated from canine faeces. After confirming by conventional and then by 16S rRNA sequencing, the identified strain RW1 was in vitro screened for its survivability in simulated gastrointestinal conditions, low pH, bile salts and adhesion to gut epithelial tissues, growth inhibitory effects on common pathogens and anti-inflammatory potential by measuring the mRNA expression level of IL-6, IL-8, IL-1β in Salmonella-infected MODE-K cells. Furthermore, the effects on epithelial barrier function and host defensin peptide (beta-defensin 3) was studied by measuring the mRNA expression level of tight junction protein (occludin) and beta-defensin 3 in MODE-K cells. The strain RW1 showed a considerable potential to survive in simulated gastrointestinal environmental conditions, low pH and high bile salt concentrations along with good adhesion to MODE-K cell line. Pathogenic bacterial growth and their adhesion to MODE-K cell line were significantly inhibited by the strain RW1. Real-time PCR analyses demonstrated that the strain RW1 inhibited Salmonella-induced pro-inflammatory cytokines (IL-6, IL-8 and IL-1β) production and reinforced the expression of tight junction protein (occludin). The strain RW1 did not induce mRNA expression of beta-defensin 3. Based on in vitro results, the strain RW1 has the potential to be used as a probiotic supplement in dogs. However, further study involving in vivo health effects is needed. Antibiotics have many side effects and nowadays the probiotics are considered as a potential alternative to antibiotics. This study evaluates the probiotic potential of dog isolated L. plantarum strain RW1 to use it as a dietary supplement in dogs feeding to control infectious diseases.

Cytoprotective Effects of Lactobacilli on Mouse Epithelial Cells during Salmonella Infection

Treatment of common pathogens, such as Salmonella species, Escherichia coli, Staphylococcus aureus, etc., is a big challenge for a practitioner. Antibiotics’ side effects during their application for the treatment of infectious diseases should not be underestimated as they have many issues, such as the transfer of antibiotics-resistant genes, dysbiosis, and antibiotic-resistant strains, which is the main hurdle in the eradication of diseases. To avoid these antibiotics complications, in modern countries, the interest of using probiotics in feed supplementation to promote health and prevent or treat intestinal infectious diseases has been increasing. The purpose of the present study was to evaluate the probiotic potential of three Lactobacilli strains isolated from clinically healthy dogs for their further utilization as a dietary supplement for dogs to avoid pathogenic and antibiotic complication. After 16SrRNA sequencing, in vitro tests were conducted to assess the survival potential of Lactobacilli under simulated gastrointestinal conditions and adhesion ability to the MODE-K cell line, effects on epithelial barrier function, anti-inflammatory activities, effects on host defensin peptides (beta-defensin 3), and inhibitory effects on common pathogens. Lactobacilli showed considerable potential to survive in simulated gastrointestinal environmental conditions, low pH, and high bile salt concentrations along with good adhesion properties with MODE-K cells. Pathogenic bacterial growth and their adhesion to MODE-K cells were significantly inhibited by Lactobacilli. Real-time PCR analyses further demonstrated that the L. acidophilus strain AR1 and AR3 inhibit Salmonella-induced proinflammatory cytokine (IL-6, IL-8, IL-1β) production and reinforce the expression of tight junction protein (occludin). None of the strains induce mRNA expression of beta-defensin 3 in MODE-K cells. Based on the in vitro results, the L. acidophilus strain AR1 has the potential to be supplemented in canine feed. However, further in vivo studies investigating health-promoting effects are awaited.

Implications of Human Antimicrobial Peptide Defensin Beta-1 in Clinical Oral Squamous Cell Carcinoma Patients via an Integrated Bioinformatics Approach.

Background The human antimicrobial peptide defensin beta 1 (DEFB1) has been found to play antimicrobial and anti-inflammatory roles in oral diseases; however, its tumor-regulating role in oral squamous cell carcinoma (OSCC) has not yet been researched by using an integrative bioinformatics approach. Objective To investigate the regulating mechanisms of the DEFB1 gene in OSCC in terms of its expression patterns, prognostic values, biological functions, and implication for tumor immunity. Methods The DEFB1 gene expression pattern and regulatory involvement in OSCC were investigated using publically accessible data from TCGA database. R software tools and public web servers were utilized to conduct statistical analysis of data from cancer and noncancerous samples. Results DEFB1 was found to be significantly downregulated in OSCC tumor samples compared with healthy control oral samples. The DEFB1 gene was found associated with the prognostic outcomes of OSCC, and its upregulation represented better survival outcome. Gene set enrichment analysis (GSEA) results showed that DEFB1-significantly correlated genes were mainly enriched in four signaling pathways mediating the antitumor role of DEFB1 in OSCC, including extracellular matrix-related pathway, RTK/PI3K/AKT/mTOR pathway, keratinization, and cytokine-related pathway. The gene-gene interaction network showed that DEFB1 was closely correlated with several genes, for example, CCR6 (C-C motif chemokine receptor 6), CXCL1 (C-X-C motif chemokine ligand 1), MAP4K2 (mitogen-activated protein kinase kinase kinase kinase 2), PTGER3 (prostaglandin E receptor 3), and MMP7 (matrix metallopeptidase 7). Moreover, DEFB1 was found to be involved in the tumor immunity of OSCC by regulating the function of tumor macrophage cells, mast cells, T cells, and NK cells. Conclusions Given the dysregulation, prognostic value, and tumor progression-related biological pathway alteration, indicating the tumor immune-modulatory role of DEFB1 in OSCC, the DEFB1 gene should be regarded as a potential therapeutic target for treating oral cancer.

New Pathophysiology Insight of Gut Microbiota’s Influence on Systemic Lupus Erythematosus: A Brief Review

Systemic lupus erythematosus (SLE) is a commonly found chronic autoimmune disease that affects multiple organs and systems. According to a new study, commensal gut microbiota promotes chronic autoimmunity in SLE patients with a lower Firmicutes/Bacteroidetes ratio. It has to do with the activation of inflammatory mediators such as toll-like receptors (TLRs), NOD-like receptors, adaptive immune response, and antimicrobial peptides mucins, defensins, and immunoglobulin A production. Aside from that, a decreased Firmicutes/Bacteroidetes ratio may cause T helper cells and regulatory T cells to become activated, both of which are linked to autoimmune activation in SLE. Theoretically, the immunomodulatory influence of food components in immune system activation has been the focus of nutrition for SLE patients, but probiotics and prebiotics have also been shown to dramatically enhance gut microbiota associated to SLE.

Phlebotomus papatasi Antimicrobial Peptides in Larvae and Females and a Gut-Specific Defensin Upregulated by Leishmania major Infection.

Phlebotomus papatasi is the vector of Leishmania major, causing cutaneous leishmaniasis in the Old World. We investigated whether P. papatasi immunity genes were expressed toward L. major, commensal gut microbes, or a combination of both. We focused on sand fly transcription factors dorsal and relish and antimicrobial peptides (AMPs) attacin and defensin and assessed their relative gene expression by qPCR. Sand fly larvae were fed food with different bacterial loads. Relish and AMPs gene expressions were higher in L3 and early L4 larval instars, while bacteria 16S rRNA increased in late L4 larval instar, all fed rich-microbe food compared to the control group fed autoclaved food. Sand fly females were treated with an antibiotic cocktail to deplete gut bacteria and were experimentally infected by Leishmania. Compared to non-infected females, dorsal and defensin were upregulated at early and late infection stages, respectively. An earlier increase of defensin was observed in infected females when bacteria recolonized the gut after the removal of antibiotics. Interestingly, this defensin gene expression occurred specifically in midguts but not in other tissues of females and larvae. A gut-specific defensin gene upregulated by L. major infection, in combination with gut-bacteria, is a promising molecular target for parasite control strategies.

Effect of food source availability in the salivary gland transcriptome of the unique burying beetle Nicrophorus pustulatus (Coleoptera: Silphidae).

Nicrophorus is a genus of beetles that bury and transform small vertebrate carcasses into a brood ball coated with their oral and anal secretions to prevent decay and that will serve as a food source for their young. Nicrophorus pustulatus is an unusual species with the ability to overtake brood of other burying beetles and whose secretions, unlike other Nicrophorus species, has been reported not to exhibit antimicrobial properties. This work aims to better understand how the presence or absence of a food source influences the expression of genes involved in the feeding process of N. pustulatus. To achieve that, total RNA was extracted from pooled samples of salivary gland tissue from N. pustulatus and sequenced using an Illumina platform. The resulting reads were used to assemble a de novo transcriptome using Trinity. Duplicates with more than 95% similarity were removed to obtain a “unigene” set. Annotation of the unigene set was done using the Trinotate pipeline. Transcript abundance was determined using Kallisto and differential gene expression analysis was performed using edgeR. A total of 651 genes were found to be differentially expressed, including 390 upregulated and 261 downregulated genes in fed insects compared to starved. Several genes upregulated in fed beetles are associated with the insect immune response and detoxification processes with only one transcript encoding for the antimicrobial peptide (AMP) defensin. These results confirm that N. pustulatus does not upregulate the production of genes encoding AMPs during feeding. This study provides a snapshot of the changes in gene expression in the salivary glands of N. pustulatus following feeding while providing a well described transcriptome for the further analysis of this unique burying beetle.

Neutrophil-Specific Defensin Receptors Prevent Skin Dysbiosis and Bacterial Infection

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that epithelial cell-derived antimicrobial peptides defensins activate orphan GPCRs Mrgpra2a/b on neutrophils. This signaling axis is required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Furthermore, we found that defensins and Mrgpra2 are critical for combatting S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggers neutrophils to release the pro-inflammatory cytokine IL-1β, which is vital for proper amplification and propagation of the antibacterial immune response. This study demonstrates the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Transcriptome Profiling Reveals a Novel Mechanism of Antiviral Immunity Upon Sacbrood Virus Infection in Honey Bee Larvae (Apis cerana).

The honey bee is one of the most important pollinators in the agricultural system and is responsible for pollinating a third of all food we eat. Sacbrood virus (SBV) is a member of the virus family Iflaviridae and affects honey bee larvae and causes particularly devastating disease in the Asian honey bees, Apis cerana. Chinese Sacbrood virus (CSBV) is a geographic strain of SBV identified in China and has resulted in mass death of honey bees in China in recent years. However, the molecular mechanism underlying SBV infection in the Asian honey bee has remained unelucidated. In this present study, we employed high throughput next-generation sequencing technology to study the host transcriptional responses to CSBV infection in A. cerana larvae, and were able to identify genome-wide differentially expressed genes associated with the viral infection. Our study identified 2,534 differentially expressed genes (DEGs) involved in host innate immunity including Toll and immune deficiency (IMD) pathways, RNA interference (RNAi) pathway, endocytosis, etc. Notably, the expression of genes encoding antimicrobial peptides (abaecin, apidaecin, hymenoptaecin, and defensin) and core components of RNAi such as Dicer-like and Ago2 were found to be significantly upregulated in CSBV infected larvae. Most importantly, the expression of Sirtuin target genes, a family of signaling proteins involved in metabolic regulation, apoptosis, and intracellular signaling was found to be changed, providing the first evidence of the involvement of Sirtuin signaling pathway in insects’ immune response to a virus infection. The results obtained from this study provide novel insights into the molecular mechanism and immune responses involved in CSBV infection, which in turn will contribute to the development of diagnostics and treatment for the diseases in honey bees.

Microbiota profiling in aging-associated inflammation and liver degeneration

BackgroundThe number of people above the age of 60 years is raising world-wide being associated with an increase in the prevalence of aging-associated impairments and even diseases. Recent studies suggest that aging is associated with alterations in bacterial endotoxin levels and that these changes may add to low-grade inflammation, the so-called ‘inflammaging’, and aging-associated liver degeneration. However, mechanisms involved, and especially, the interaction of intestinal microbiota and barrier in the development of aging-associated inflammation and liver degeneration have not been fully understood. ObjectiveThe aim of the present study was to determine if intestinal microbiota composition changes with age and if these alterations are associated with changes of markers of intestinal barrier function and the development of inflammation and liver degeneration. MethodsBlood, liver, small and large intestinal tissue of male 2-, 15-, 24- and 30-months old C57BL/6 mice fed standard chow were obtained. Intestinal microbiota composition, expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function were measured. Furthermore, indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissue were assessed. ResultsPairwise comparisons of the microbial community in the small intestine showed differences between 2- and 24-, 15- and 24-, as well as 15- and 30-months old animals while Shannon’s diversity, species richness and evenness indexes did not differ in both small and large intestine, respectively, between age groups. Concentrations of nitric oxide were significantly lower in small intestine of 15-, 24- and 30-months old mice compared to 2-months old mice while mRNA expression of the antimicrobial peptides defensin alpha 1 and lysozyme 1 was unchanged. In contrast, in liver tissue, older age of animals was associated with increasing inflammation and the development of fibrosis in 24- and 30-months old mice. Numbers of inflammatory foci and neutrophils in livers of 24- and 30-months old mice were significantly higher compared to 2-months old mice. These alterations were also associated with higher endotoxin levels in plasma as well as an increased mRNA expression of Lbp and Tlr1, Tlr2, Tlr4, Tlr6 and Tlr9 in livers in older mice. ConclusionDespite no consistent and robust changes of microbiota composition in small and/or large intestine of mice of different age were observed, our data suggest that alterations of markers of intestinal barrier function in small intestine are associated with an induction of several Tlrs and beginning hepatic inflammation in older mice and increase with age.

Inhibition of defensin A and cecropin A responses to dengue virus 1 infection in Aedes aegypti.

IntroductionIt is essential to determine the interactions between viruses and mosquitoes to diminish dengue viral transmission. These interactions constitute a very complex system of highly regulated pathways known as the innate immune system of the mosquito, which produces antimicrobial peptides that act as effector molecules against bacterial and fungal infections. There is less information about such effects on virus infections.ObjectiveTo determine the expression of two antimicrobial peptide genes, defensin A and cecropin A, in Aedes aegypti mosquitoes infected with DENV-1.Materials and methodsWe used the F1 generation of mosquitoes orally infected with DENV-1 and real-time PCR analysis to determine whether the defensin A and cecropin A genes played a role in controlling DENV-1 replication in Ae. aegypti. As a reference, we conducted similar experiments with the bacteria Escherichia coli.ResultsBasal levels of defensin A and cecropin A mRNA were expressed in uninfected mosquitoes at different times post-blood feeding. The infected mosquitoes experienced reduced expression of these mRNA by at least eightfold when compared to uninfected control mosquitoes at all times post-infection. In contrast with the behavior of DENV-1, results showed that bacterial infection produced up-regulation of defensin and cecropin genes; however, the induction of transcripts occurred at later times (15 days). Conclusion: DENV-1 virus inhibited the expression of defensin A and cecropin A genes in a wild Ae. aegypti population from Venezuela.

Molecular characterization of diverse defensins (γ-thionins) from Capsicum annuum flowers and their effects on the insect pest Helicoverpa armigera

Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) constituting the host defense system. They are known to interact with the cell membranes to exhibit antifungal and antibacterial activity. Though defensin genes are inducible under biotic and abiotic stress, their involvement in insect herbivore defense is not well studied. In this work we studied the phylogeny, structural diversity, biochemical and functional properties of defensins from Capsicum annuum L with particular emphasis on their interaction with insect and fungal pathogens. Phylogenetic analysis of defensin peptides from Solanaceae, Pinaceae, Fabaceae, Asteraceae, Brassicaceae and similar peptides from invertebrates divided them into ten groups and Capsicum peptides were represented in most of them. Three thionin (CanThio-1, −2, −3) and two defensin (CanDef-20, −21) genes isolated from the flowers of Capsicum annuum were cloned and recombinant proteins were expressed. The mature peptide amino acid sequences of CanThio and CanDef were 54–75 aa with 21.82 to 73.81% similarity. They contained the conserved features of defensin peptides like the gamma core region and the presence of eight cysteine residues. Predicted structural alignment indicated that CanThio and CanDef had structural diversity and structurally unaligned loop regions.The recombinant CanThio and CanDef inhibited 5% - 65% Aspergillus oryzae amylase activity at 20 μM and ~ 20% of Helicoverpa armigera gut amylase activity at 50 μM peptide concentrations. CanThio-2 displayed bovine trypsin inhibition (~14%) while all the CanThio and CanDef inhibited ~20% of H. armigera gut proteinase activity at 20 μM. H. armigera larvae fed on an artificial diet incorporated with recombinant CanThio-2 and CanDef-20 (125 μg/ml) exhibited a 15% reduction in larval mass and 13% reduction in pupal mass. The CanThio and CanDef peptides also exhibited antifungal properties against pathogenic fungus Fusarium oxysporum at 5 μg/ml, where CanThio-2 and CanDef-20 showed the strongest activity, which may be attributed to the positively charged amino acids in the γ-core region.We conclude that despite having relatively similar conserved three-dimensional structures CanThio and CanDef display functional variability, which can be utilized for enhancing insect and fungal resistance in plants.

Identify immune-related genes of adzuki bean weevil (Callosobruchus chinensis) in response to bacteria challenge by transcriptome analysis

BackgroundCallosobruchus chinensis is one of the important postharvest pests in legume growing areas. Bacterial pesticide is a potential alternative method to control storage pests. However, the effect of these pathogen bacteria on storage pests, and the molecular mechanisms of insect response remain to be to investigated. ResultsUsing the next generation sequencing technology, we established a transcriptomic library for C. chinensis larvae in response to Escherichia coli. Total of 355 differential expressed genes (DEGs) were identified, which 178 DEGs were upregulated, and 177 DEGs were downregulated compared to control group. To validate the RNA-seq analysis, 20 DEGs and 14 immune-related genes were selected to perform quantitative polymerase chain reaction (RT-qPCR). These immune-related genes were involved in recognition (peptidoglycan recognition proteins), signal transduction (fibrinogen-related proteins, serine proteinases and NF-κB), and execution effectors (phenoloxidase, defensin, attacin, and antimicrobial peptide). In addition, genes that encode digestive and respiratory enzymes were altered in C. chinensis larvae in response to infection. Some genes that involved in juvenile hormone and insulin pathway appeared to express differentially, suggesting that pathogen infection might lead to developmental arrest. Furthermore, iron homeostasis and chitin metabolism appeared significantly altered after infection. ConclusionIn this study, we characterized the immune response of C. chinensis larvae in response to E. coli using RNA-seq, from pathogen recognition, signal transduction, to execution. Some other identified genes were involved in iron homeostasis, respiration, and digestion. A better understanding of molecular response of beetle to pathogen will facilitate us to develop an available strategy to control storage pests.

The seven constitutive respiratory defense barriers against SARS-CoV-2 infection.

Before eliciting an adaptive immune response, SARS-CoV-2 must overcome seven constitutive respiratory defense barriers. The first is the mucus covering the respiratory tract’s luminal surface, which entraps inhaled particles, including infectious agents, and eliminates them by mucociliary clearance. The second barrier comprises various components present in the airway lining fluid, the surfactants. Besides providing low surface tension that allows efficient gas exchange at the alveoli, surfactants inhibit the invasion of epithelial cells by respiratory viruses, enhance pathogen uptake by phagocytes, and regulate immune cells’ functions. The respiratory tract microbiota constitutes the third defense barrier against SARS-CoV-2. It activates the innate and adaptive immune cells and elicits anti-infectious molecules such as secretory IgA antibodies, defensins, and interferons. The fourth defense barrier comprises the antimicrobial peptides defensins, and lactoferrin. They show direct antiviral activity, inhibit viral fusion, and modulate the innate and adaptive immune responses. Secretory IgA antibodies, the fifth defense barrier, besides protecting the local microbiota against noxious agents, also inhibit SARS-CoV-2 cell invasion. If the virus overcomes this barrier, it reaches its target, the respiratory epithelial cells. However, these cells also act as a defense barrier, the sixth one, since they hinder the virus’ access to receptors and produce antiviral and immunomodulatory molecules such as interferons, lactoferrin, and defensins. Finally, the sensing of the virus by the cells of innate immunity, the last constitutive defense barrier, elicits a cascade of signals that activate adaptive immune cells and may inhibit the development of productive infection. The subject of the present essay is discussing these mechanisms.

Study on the Inhibitory Activity of a Synthetic Defensin Derived from Barley Endosperm against Common Food Spoilage Yeast.

In the food industry, food spoilage is a real issue that can lead to a significant amount of waste. Although current preservation techniques are being applied to reduce the occurrence of spoilage microorganisms, the problem persists. Food spoilage yeast are part of this dilemma, with common spoilers such as Zygosaccharomyces, Kluyveromyces, Debaryomyces and Saccharomyces frequently encountered. Antimicrobial peptides derived from plants have risen in popularity due to their ability to reduce spoilage. This study examines the potential application of a synthetic defensin peptide derived from barley endosperm. Its inhibitory effect against common spoilage yeasts, its mechanisms of action (membrane permeabilisation and overproduction of reactive oxygen species), and its stability in different conditions were characterised. The safety of the peptide was evaluated through a haemolysis and cytotoxicity assay, and no adverse effects were found. Both assays were performed to understand the effect of the peptide if it were to be consumed. Its ability to be degraded by a digestive enzyme was also examined for its safety. Finally, the peptide was successfully applied to different beverages and maintained the same inhibitory effects in apple juice as was observed in the antiyeast assays, providing further support for its application in food preservation.

Utilization of a recombinant defensin from Maize (Zea mays L.) as a potential antimicrobial peptide

The search for effective and bioactive antimicrobial molecules to encounter the medical need for new antibiotics is an encouraging area of research. Plant defensins are small cationic, cysteine-rich peptides with a stabilized tertiary structure by disulfide-bridges and characterized by a wide range of biological functions. The heterologous expression of Egyptian maize defensin (MzDef) in Escherichia coli and subsequent purification by glutathione affinity chromatography yielded 2 mg/L of recombinant defensin peptide. The glutathione-S-transferase (GST)-tagged MzDef of approximately 30 kDa in size (26 KDa GST + ~ 4 KDa MzDef peptide) was immunodetected with anti-GST antibodies. The GST-tag was successfully cleaved from the MzDef peptide by thrombin, and the removal was validated by the Tris-Tricine gel electrophoresis. The MzDef induced strong growth inhibition of Rhizoctonia solani, Fusarium verticillioides, and Aspergillus niger by 94.23%, 93.34%, and 86.25%, respectively, whereas relatively weak growth inhibitory activity of 35.42% against Fusarium solani was recorded. Moreover, strong antibacterial activities were demonstrated against E. coli and Bacillus cereus and the moderate activities against Salmonella enterica and Staphylococcus aureus at all tested concentrations (0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 µM). Furthermore, the in vitro MTT assay exhibited promising anticancer activity against all tested cell lines (hepatocellular carcinoma, mammary gland breast cancer, and colorectal carcinoma colon cancer) with IC50 values ranging from 14.85 to 29.85 µg/mL. These results suggest that the recombinant peptide MzDef may serve as a potential alternative antimicrobial and anticancer agent to be used in medicinal application.