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[Display omitted] • Immunized Locusta migratoria transcriptome was de novo assembled. • Decoded Locusta migratoria antimicrobial peptide from its transcriptome. • Curated antimicrobial peptides pass molecular and aggregation filters. • Two synthesized Locusta migratoria peptides showed antimicrobial activity. Evolutionary immune system upgradation is done by all living organism to survive from the invading pathogens. Insects has a strong defense system comprising of anatomical barrier, humoral antimicrobial peptide (AMP) production and cellular immune components. Locusta migratoria (L. migratoria) is an agricultural pest insect migrating long distance might encounter distinct pathogens. Owing to this, we aimed in identifying AMPs present in the L. migratoria and elucidate the antimicrobial activity. The migratory locust was immunized with Escherichia coli, Staphylococcus aureus, Candida albicans mixture, then RNA was isolated and sequenced. The L. migratoria transcriptome was de novo assembled using trinity and screened for AMPs propensity specifically molecular and aggregation properties. The novelty is tested by blasting in known AMP databases. Finally, obtained novel putative AMPs were then tested for antimicrobial and hemolytic activity. The prediction resulted in 3,524 putative AMPs which was further screened down to ten AMPs for final testing. Two peptides showing promising antimicrobial effects were obtained. All the analyzed peptides showed no hemolysis confirms its non-toxicity. Thus, our peptides could be promising drug candidate and can be used as an alternative to antibacterial or antifungal therapy with further validations. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is a potentially powerful weapon against Candida albicans, and the regulation of intracellular NO levels is therefore important for controlling its physiological functions. Lactoferricin B like peptide (LBLP) is a 23‐mer antimicrobial peptide (AMP) derived from the Scolopendra subspinipes mutilans. We confirmed that LBLP treatment led to the generation of endogenous NO in C. albicans, which was associated with the NO synthase pathway. Here, we examined the antifungal activity of LBLP with focus on intracellular NO. Total glutathione levels were measured to evaluate cellular defense capacity against NO. LBLP decreased total glutathione levels, leading to nitrosative stress. LBLP also inhibited mitochondrial respiration and altered the NAD+/NADH ratios. Inhibition of mitochondrial respiration induced mitochondrial membrane depolarization, thus leading to calcium homeostasis disruption and mitochondrial superoxide anion accumulation. Consequently, treatment of C. albicans with LBLP resulted in apoptosis. These physiological changes were attenuated when NO generation was inhibited. Our data strongly indicate that LBLP mediates apoptosis by affecting intracellular NO homeostasis. These results on antifungal activity of LBLP and its mechanism indicate the therapeutic promise of this AMP and support the role of NO in cell death regulation. [ABSTRACT FROM AUTHOR]

A novel antimicrobial peptide (AMP) was identified from the centipede Scolopendra subspinipes mutilans by RNA sequencing, and the amino acid sequences predicted from the sequenced mRNAs were compared with those of known AMPs. We named this peptide scolopendin, according to its origin, and investigated the molecular mechanisms underlying its antimicrobial activity. Our findings showed that scolopendin had antimicrobial activity against several pathogenic microorganisms, but did not produce hemolysis of human erythrocytes. In addition, disturbances in the cell membrane potential, induction of potassium release from the cytosol, and increased membrane permeability of the microbes Candida albicans and Escherichia coli O157 were detected by the use of 3,3′-dipropylthiacarbocyanine iodide [DiSC 3 (5)] dye, potassium leakage assay, and propidium iodide influx assay, respectively, following scolopendin treatment. Further evidence to support the membrane-targeted action of scolopendin was obtained using artificial liposomes as models of the cell membrane. Use of calcein and FITC-labeled dextran leakage assays from scolopendin-treated giant unilamellar vesicles and large unilamellar vesicles showed that scolopendin has a pore-forming action on microbial membrane, with an estimated pore radius of 2.3–3.3 nm. In conclusion, scolopendin is a novel and potent AMP with a membrane-targeted mechanism of action. [ABSTRACT FROM AUTHOR]

Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans . We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans , resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3′-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane. [ABSTRACT FROM AUTHOR]

Abstract: The centipede Scolopendra subspinipes mutilans has been a medically important arthropod species by using it as a traditional medicine for the treatment of various diseases. In this study, we derived a novel lactoferricin B like peptide (LBLP) from the whole bodies of adult centipedes, S. s. mutilans, and investigated the antifungal effect of LBLP. LBLP exerted an antifungal and fungicidal activity without hemolysis. To investigate the antifungal mechanism of LBLP, a membrane study with propidium iodide was first conducted against Candida albicans. The result showed that LBLP caused fungal membrane permeabilization. The assays of the three dimensional flow cytometric contour plot and membrane potential further showed cell shrinkage and membrane depolarization by the membrane damage. Finally, we confirmed the membrane-active mechanism of LBLP by synthesizing model membranes, calcein and FITC-dextran loaded large unilamellar vesicles. These results showed that the antifungal effect of LBLP on membrane was due to the formation of pores with radii between 0.74nm and 1.4nm. In conclusion, this study suggests that LBLP exerts a potent antifungal activity by pore formation in the membrane, eventually leading to fungal cell death. [Copyright &y& Elsevier]

Highlights: [•] l- and d-CopA3 was designed by replacing an amino acid of CopN5 derived from coprisin. [•] l- and d-CopA3 exerted potent antifungal activity without hemolysis. [•] l- and d-CopA3 had membrane permeabilization mechanism in the fungal cells. [•] Only d-CopA3 maintained a potent antifungal activity under proteolytic condition. [•] d-CopA3 has potential as a novel antimicrobial agent. [Copyright &y& Elsevier]

Abstract: Papiliocin is a 37-residue peptide isolated from the swallowtail butterfly Papilio xuthus. In this study, we found that Papiliocin induced the accumulation of reactive oxygen species (ROS) and hydroxyl radicals known to be important regulators of apoptosis in Candida albicans. To examine the relationship between the accumulation of ROS and the induction of apoptosis, we investigated the apoptotic effects of Papiliocin using apoptotic markers. Cells treated with Papiliocin showed a series of cellular changes normally seen in cells undergoing apoptosis: plasma membrane translocation of phosphatidylserine from the inner to the outer membrane leaflet, measured by Annexin V staining, dissipation of the mitochondrial membrane potential, observed by DiOC6(3) staining; and the presence of active metacaspases, measured using the CaspACE FITC-VAD-FMK, as early apoptotic events. In addition, DNA condensation and fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. Therefore, these results suggest that Papiliocin leads to apoptosis in C. albicans via ROS accumulation. [Copyright &y& Elsevier]

Abstract: Previously, the antimicrobial effects and membrane-active action of psacotheasin in Candida albicans were investigated. In this study, we have further found that a series of characteristic cellular changes of apoptosis in C. albicans can be induced by the accumulation of intracellular reactive oxygen species, specifically hydroxyl radicals, the well-known important regulators of apoptosis. Cells treated with psacotheasin showed diagnostic markers in yeast apoptosis at early stages: phosphatidylserine externalization from the inner to the outer membrane surface, visualized by Annexin V-staining; mitochondrial membrane depolarization, observed by DiOC6(3) staining; and increase of metacaspase activity, measured using the CaspACE FITC-VAD-FMK. Moreover, DNA fragmentation and condensation also revealed apoptotic phenomena at late stages through the TUNEL assay staining and DAPI staining, respectively. Taken together, our findings suggest that psacotheasin possess an antifungal property in C. albicans via apoptosis as another mode of action. [Copyright &y& Elsevier]

Simple Summary: Multidrug resistance for classical antibiotics is the major problem faced in clinical infections. Insects have antimicrobial peptides (AMPs) which are a part of the immune repertoire to protect it from microbial pathogens. AMPs are short chain protein molecules that function as antibacterial, antifungal, antiparasitic and antiviral factors. Insect AMPs are present in their hemolymph or produced in response to entry of pathogens and kill them to protect the host. The objective of this study is to identify novel AMPs from Psacothea hilaris, the yellow spotted long horned beetle. The beetle was immunized with bacteria and fungi and RNA was isolated. The RNA was processed and screened by in silico strategies to identify novel AMPs. We obtained one potential candidate which was tested to be effective against harmful bacteria and fungi. This will be useful in treating multidrug resistant microbes alone or in combination with antibiotics with future validations. Antimicrobial peptides (AMPs) are the frontline innate defense system evolutionarily preserved in insects to combat invading pathogens. These AMPs could serve as an alternative to classical antibiotics to overcome the burden of treating multidrug resistant bacteria. Psacotheasin, a knottin type AMP was isolated from Psacothea hilaris and shown to exhibit antimicrobial activity, especially against fungi through apoptosis mediated cell death. In this study, we aimed to identify novel probable AMPs from Psacothea hilaris, the yellow spotted longicorn beetle. The beetle was immunized with the two bacterial strains (E. coli and S. aureus), and the yeast strain C. albicans. After immunization, total RNA was isolated and sequenced in Illumina platform. Then, beetle transcriptome was de novo assembled and searched for putative AMPs with the known physiochemical features of the AMPs. A selection of AMP candidates were synthesized and tested for antimicrobial activity. Four peptides showed stronger activity against E. coli than the control AMP, melittin while one peptide showed similar activity against S. aureus. Moreover, four peptides and two peptides showed antifungal activity stronger than and similar to melittin, respectively. Collectively one peptide showed both antibacterial and antifungal activity superior to melittin; thus, it provides a potent antimicrobial peptide. All the peptides showed no hemolysis in all the tested concentrations. These results suggest that in silico mining of insects' transcriptome could be a promising tool to obtain and optimize novel AMPs for human needs. [ABSTRACT FROM AUTHOR]

In this study, we identified scolopendrasin X, a novel antimicrobial peptide (AMP), from centipede Scolopendra subspinipes mutilans . Scolopendrasin X strongly stimulated mouse neutrophils, resulting in intracellular calcium increase, chemotactic migration through pertussis toxin-sensitive G-protein and phospholipase C pathway, and increased superoxide anion production in neutrophils. Target receptor for scolopendrasin X, formyl peptide receptor (FPR)2 mediated scolopendrasin X-induced neutrophil activation. Moreover, scolopendrasin X significantly blocked inflammatory cytokine production induced by lipopolysaccharide in mouse neutrophils. Taken together, our results suggest that the novel AMP scolopendrasin X can be used as a material to regulate neutrophil activity through FPR2. [ABSTRACT FROM AUTHOR]

Recently, we reported that Lumbricusin, an antimicrobial peptide isolated from earthworm Lumbricus terrestris , enhanced neuronal proliferation and ameliorated motor dysfunction and dopaminergic neurodegeneration. Accumulating evidence suggests that neurodegeneration is the primary pathological feature of acute or chronic inflammation mediated by microglia, the resident macrophage of the central nervous system. Therefore, microglial activation inhibitors may be useful as therapeutic agents for neurodegenerative diseases. To determine whether Lumbricusin ameliorates neuroinflammation through inhibition of microglial activation by lipopolysaccharides (LPS), we newly synthesized 9-mer Lumbricusin analogues based on the amino acid sequence of Lumbricusin. One of these, Lumbricusin Analogue 5 (LumA5; QLICWRRFR-NH 2 ), markedly reduced expression of enzymes (COX-2, iNOS), cytokines (IL-6, IL-1β, TNF-α), and signal transduction factors (AKT, MAPKs, NF-κB) involved in inflammation triggered by LPS in vitro and in vivo . In addition, LumA5 inhibited the cytotoxicity of conditioned medium prepared by LPS-activated BV-2 microglia to neuronal SH-SY5Y cells and improved cell viability. These results indicate that LumA5 may be a potential therapeutic agent for the treatment of various neuroinflammatory conditions. [ABSTRACT FROM AUTHOR]

Highlights: [•] We report the finding of CopW that has a potent antimicrobial activity. [•] CopW is a new cysteine-free nonapeptide derived from coprisin. [•] CopW also exhibited significant synergistic activity with ampicillin. [Copyright &y& Elsevier]

Abstract: The novel 43-residue, insect defensin-like peptide coprisin, isolated from the dung beetle, Copris tripartitus, is a potent antibiotic with bacterial cell selectivity, exhibiting antimicrobial activities against Gram-positive and Gram-negative bacteria without exerting hemolytic activity against human erythrocytes. Tests against Staphylococcus aureus using fluorescent dye leakage and depolarization measurements showed that coprisin targets the bacterial cell membrane. To understand structure–activity relationships, we determined the three-dimensional structure of coprisin in aqueous solution by nuclear magnetic resonance spectroscopy, which showed that coprisin has an amphipathic α-helical structure from Ala19 to Arg28, and β-sheets from Gly31 to Gln35 and Val38 to Arg42. Coprisin has electropositive regions formed by Arg28, Lys29, Lys30, and Arg42 and ITC results proved that coprisin and LPS have electrostatically driven interactions. Using measurements of nitric oxide release and inflammatory cytokine production, we provide the first verification of the anti-inflammatory activity and associated mechanism of an insect defensin, demonstrating that the anti-inflammatory actions of the defensin-like peptide, coprisin, are initiated by suppressing the binding of LPS to toll-like receptor 4, and subsequently inhibiting the phosphorylation of p38 mitogen-activated protein kinase and nuclear translocation of NF-kB. In conclusion, we have demonstrated that an amphipathic helix and an electropositive surface in coprisin may play important roles in its effective interaction with bacterial cell membranes and, ultimately, in its high antibacterial activity and potent anti-inflammatory activity. In addition to elucidating the antimicrobial action of coprisin, this work may provide insight into the mechanism of action of insect defense systems. [Copyright &y& Elsevier]

• Insect antimicrobial peptides (AMPs) attracting attention as alternative foods and medicines. • The discovery of defensin through transcriptome analysis of the red-striped golden stink bug (Poecilocoris lewisi) • Transcript analysis data for P. lewisi. Insect antimicrobial peptides (AMPs) have a wide range of functions and potential applications, and have recently attracted attention as alternative foods and medicines for humans. Our study performed transcriptome analysis to explore the potential of the red-striped golden stink bug (Poecilocoris lewisi), and as a result, we have discovered new features of P. lewisi that have not been identified. Specifically, defensin found in P. lewisi is a well-known AMP and is expressed by various plants, animals and fungi for host defense. Moreover, the discovery of defensin in P. lewisi provides new research and important information. In this study, we identified AMP and related DEG in P. lewisi that are closely related to human disease and immune response. These findings will provide the basis and important information for future research on P. lewisi that has not yet been studied. [ABSTRACT FROM AUTHOR]