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This study aimed to explore the intervention effect of cecropin on the inflammatory exudation caused by coinfection with H9N2 subtype avian influenza virus (AIV) and pathogenic avian Escherichia coli (APEC), providing a reference for the effective prevention and treatment of clinical co-infections of H9N2 subtype AIV and APEC. The experiment selected 150 white-feathered broilers at 14 days old, randomly divided into five groups, with three replicates per group and 10 chickens per replicate. The control group was administered 0.2 mL of sterile saline nasally. The APEC infection group was administered 0.2 mL of broth containing 2.09×109 CFU of APEC O2 strain nasally. The H9N2 infection group was administered 0.2 mL of H9N2 viral allantoic fluid (approximately 105 EID50 H9N2 virus) nasally. The H9N2 + APEC infection group was administered 0.2 mL of the pellet from the centrifugation of APEC O2 broth mixed with 0.2 mL of H9N2 viral allantoic fluid nasally. The H9N2 + APEC intervention group was treated in the same way as the H9N2 + APEC infection group and fed with cecropin in drinking water (300 mg/kg) + basic feed. The rest of the groups were fed with normal water + basic feed. The formal experimental period was 21 days. The results showed that the H9N2 + APEC infection group had very obvious clinical symptoms and a significantly increased mortality rate (P<0.05), with the most severe tracheal and pulmonary lesions. Compared with the H9N2 + APEC infection group, the H9N2 virus copy number and APEC load on the 3rd, 7th, 14th, and 21st days after infection were significantly reduced in the H9N2 infection group and the APEC infection group (P<0.0 5), the H9N2 + APEC infection group, the H9N2 virus copy number on the 3rd, 7th, 14th, and 21st days after infection was significantly reduced (P<0.05), and the APEC load on the 7th, 14th, and 21st days after infection was significantly reduced (P<0.05). The study showed that cecropin can significantly alleviate respiratory symptoms caused by H9N2 subtype AIV, APEC, and their co-infection, and reduce mortality rates. Its mechanism is related to the reduction of pathogen load in tissues and the repair of the respiratory system, improving respiratory function. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) in insects are short peptides that play an important role in their innate defense system. The production of AMPs is typically induced by an infection of pathogenic microorganisms, but cold stress may also cause upregulation of insect AMP genes. However, little is known about the functions of AMPs in cold stress situations and the recovery afterwards. As most important results, in this project with Lasioderma serricorne that can tolerate long storage under cold conditions, we identified and investigated four AMP genes (LsAtt1, LsAtt3, LsCec and LsDef-like). We confirmed their anti-bactericide activity in in vitro assays and in RNAi assays. The cross treatments with cold stress showed that LsDef-like was upregulated under cold stress and its silencing caused a loss of survival. In contrast, the expression of the other three AMPs did not change, and their silencing prolonged the recovery time. We believe that our cross treatments, revealing an association between immune activation and cold stress with AMPs, will increase our basic understanding of the immune system and the “cross-talk” with cold resistance. [ABSTRACT FROM AUTHOR]

China Antimicrobial peptides could inhibit the growth of harmful bacteria and promote the growth performance in weaned piglets. Here, we investigated the effects of dietary supplementation with cecropin antimicrobial peptides (CAP) on growth performance, diarrhea rate, intestinal health in nursery Hainan piglets. For this, 120 healthy nursery Hainan male piglets (13.29 ± 0.29 kg, 44 days old) were randomly divided into 5 groups--a control (CON) group (fed a basal diet), an antibiotic control (AC) group (fed a basal diet supplemented with 250 mg/kg colistin sulfate); and 3 experimental groups (provided the basal diet supplemented with 250, 500, or 1,000 mg/kg CAP). Pre-feeding lasted 7 days and the official period lasted 40 days. The results showed that compared with the CON group, dietary supplementation of 500 mg/kg CAP had significantly increased the average daily gain (ADG, p < 0.05), while the feed conversion ratio (FCR) and diarrhea rate were markedly reduced (p < 0.05), serum total protein (TP), albumin, IgA, IgM, and globulin concentrations were significantly increased (p < 0.05), where serum aspartate aminotransferase (AST) level was significantly reduced (p < 0.05), and it also increased the villus height and the villus height-to-crypt depth ratio in the jejunum, reduced the serum D-lactic acid concentrations and diamine oxidase activity, and increased the expression level of ZO-1 and occludin in the jejunum and ileum (p < 0.05), the relative abundance of Firmicutes, Lactobacillus, and Limoslactobacillus in the colon were increased (p < 0.05), whereas that of Streptococcus and Escherichia-Shigella were reduced (p < 0.05). These results indicated that dietary supplementation with 500 mg/kg CAP could improve the growth performance, reduce the diarrhea rate, improve the serum immunity, intestinal health of nursery pigs. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) show promise in enhancing resistance against pathogens. Previously, we integrated two AMP genes, cathelicidin (Cath) from alligator (Alligator mississippiensis or Alligator sinensis) and cecropin (Cec) from moth (Hyalophora cecropia), into the channel catfish (Ictalurus punctatus) genome. This study examines the efficacy of exogenous AMP gene integration in improving bacterial resistance in transgenic channel catfish and assesses the direct and pleiotropic effects of gene replacement/knockout on survival and growth based on insertion site. Transgenic Cath- and Cec-expressing fish exhibited similar or higher survival rates (P ​> ​0.05) compared to controls during the initial culture. Integration of the Cec transgene doubled the survival rate when challenged with Edwardsiella ictaluri, with knock-in (KI) of Cath further increasing bacterial resistance. Coupling Cec KI with mstn knockout (KO) increased survival 3-fold after E. ictaluri infection and growth by 50% at 4 months post-fertilization (mpf). However, random integration of Cec had a minimal effect on disease resistance and did not enhance growth. Random integration of Cath increased survival 2.5-fold and 4-fold against E. ictaluri and Flavobacterium covae, respectively, without affecting growth. Cath KI at the lh locus increased survival 4-fold when challenged with F. covae and reduced growth by 10% (P ​> ​0.05) at 24 mpf, whereas Cath KI coupled with mc4r KO resulted in a 2.5-fold increase in survival following F. covae infection compared with controls, and increased growth by 80% at 3 mpf. Simultaneous KI of Cath and Cec, along with KO of mc4r and mstn, increased survival 4-fold against E. ictaluri, while increasing growth by 50% at 3 mpf. Dual insertion of AMP genes yielded the greatest resistance to disease. These direct and pleiotropic effects may increase comprehension and societal acceptance of genetic engineering in aquaculture.

Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding Cecropins are strongly induced upon infection, pointing to their role in host defense. In Drosophila, four cecropin genes clustered in the genome (CecA1, CecA2, CecB, and CecC) are expressed upon infection downstream of the Toll and Imd pathways. In this study, we generated a short deletion ΔCecA-C removing the whole cecropin locus. Using the ΔCecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, we addressed the function of Cecropins in the systemic immune response. ΔCecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing ΔCecA-C in flies already lacking 10 other AMP genes revealed a role for Cecropins in defense against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that Cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, our work provides the first genetic demonstration of a role for Cecropins in insect host defense and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line (ΔAMP14) that lacks 14 immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond.

Egypt has witnessed the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, which has posed a serious healthcare challenge. The proper treatment choice for MDR-KP infections is not well determined which renders the problem more complicated, thus making the control of such infections a serious challenge for healthcare professionals. This study aims to encapsulate the cationic antimicrobial peptide; Cecropin-B (Cec-B), to increase its lifetime, drug targeting, and efficacy and study the antimicrobial effect of free and encapsulated recombinant rCec-B peptide on multidrug-resistant K. pneumoniae (MDR-KP) isolates. Fifty isolates were collected from different clinical departments at Theodore Bilharz Research Institute. Minimal inhibitory concentrations (MICs) of rCec-B against MDR-KP isolates were determined by the broth microdilution test. In addition, encapsulation of rCec-B peptide into chitosan nanoparticles and studying its bactericidal effect against MDR-KP isolates were also performed. The relative expression of efflux pump and porin coding genes (ArcrB, TolC, mtdK, and Ompk35) was detected by quantitative PCR in treated MDR-KP bacterial isolates compared to untreated isolates. Out of 60 clinical MDR isolates, 50 were MDR-KP. 60% of the isolates were XDR while 40% were MDR. rCec-B were bactericidal on 21 isolates, then these isolates were subjected to treatment using free nanocapsule in addition to the encapsulated peptide. Free capsules showed a mild cytotoxic effect on MDR-KP at the highest concentration. MIC of encapsulated rCec-B was higher than the free peptide. The expression level of genes encoding efflux and porin (ArcrB, TolC, mtdK, and Ompk35) was downregulated after treatment with encapsulated rCec-B. These findings indicate that encapsulated rCec-B is a promising candidate with potent antibacterial activities against drug-resistant K. pneumoniae. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune‐related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection‐responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune‐related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double‐stranded RNA and bacterial infection. [ABSTRACT FROM AUTHOR]

Simple Summary: Antimicrobial peptides (AMPs) are widely present in organisms, exhibiting broad-spectrum antimicrobial activity and rarely developing resistance. Currently, research on AMPs in many species is still limited, and they have great potential for exploration. In this study, a class of AMPs called Cecropin was identified from insect transcriptome using bioinformatics. Novel Cecropin genes were discovered in several insects and three of them were selected for experimental validation. These peptides possess antimicrobial activity against Gram-negative bacteria, exhibit a structure and mechanism of action similar to known Cecropins, and are non-toxic to mammalian cells. This study provides a reliable analytical method in the search for AMPs, which hold promising application prospects in diverse fields such as medicine, food, and beyond. Antibiotic resistance is a significant and growing threat to global public health. However, antimicrobial peptides (AMPs) have shown promise as they exhibit a broad spectrum of antibacterial activities with low potential for resistance development. Insects, which inhabit a wide range of environments and are incredibly diverse, remain largely unexplored as a source of novel AMPs. To address this, we conducted a screening of the representative transcriptomes from the 1000 Insect Transcriptome Evolution (1KITE) dataset, focusing on the homologous reference genes of Cecropins, the first identified AMPs in insects known for its high efficiency. Our analysis identified 108 Cecropin genes from 105 insect transcriptomes, covering all major hexapod lineages. We validated the gene sequences and synthesized mature peptides for three identified Cecropin genes. Through minimal inhibition concentration and agar diffusion assays, we confirmed that these peptides exhibited antimicrobial activities against Gram-negative bacteria. Similar to the known Cecropin, the three Cecropins adopted an alpha-helical conformation in membrane-like environments, efficiently disrupting bacterial membranes through permeabilization. Importantly, none of the three Cecropins demonstrated cytotoxicity in erythrocyte hemolysis tests, suggesting their safety in real-world applications. Overall, this newly developed methodology provides a high-throughput bioinformatic pipeline for the discovery of AMP, taking advantage of the expanding genomic resources available for diverse organisms. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium responsible for severe nosocomial infections and is considered a critical pulmonary pathogen for both immunocompromised and cystic fibrosis patients. Planktonic cells of P. aeruginosa possess intrinsic and acquired resistances, inactivating several classes of conventional antibiotics. Additionally, this bacterium can grow, forming biofilms, and complex structures, further hampering the action of multiple antibiotics. Here, we report the biological properties of D-Q53 CecB, an all-D enantiomer of the silkworm natural peptide Q53 CecB. Compared to the L-variant, D-Q53 CecB was resistant to in vitro degradation by humans and P. aeruginosa elastases and showed an enhanced bactericidal activity against P. aeruginosa planktonic bacteria. D-Q53 CecB was thermostable and maintained its antimicrobial activity at high salt concentrations and in the presence of divalent cations or fetal-bovine serum, although at reduced levels. Against different types of human cells, D-Q53 CecB showed cytotoxic phenomena at concentrations several folds higher compared to those active against P. aeruginosa. When L- and D-Q53 CecB were compared for their antibiofilm properties, both peptides were active in inhibiting biofilm formation. However, the D-enantiomer was extremely effective in inducing biofilm degradation, suggesting this peptide as a favorable candidate in an anti-Pseudomonas therapy. [ABSTRACT FROM AUTHOR]

Insect proteins have been proposed for human and animal food production. Safeguarding the health status of insects in mass rearing allows to obtain high‐quality products and to avoid severe economic losses due to entomopathogens. Therefore, new strategies for preserving insect health must be implemented. Modulation of the insect immune system through the diet is one such strategy. We evaluated gene expression of two antimicrobial peptides (one defensin and one cecropin) in Hermetia illucens (L.) (Diptera: Stratiomyidae) reared on different diets. Analyses were performed on prepupae and 10‐day‐old larvae reared on cereal‐ and municipal organic waste‐based diets and on only prepupae reared on a cereal‐based diet supplemented with sunflower, corn, or soybean oil. The inclusion of sunflower oil at different points in the cereal‐based diet was also evaluated. Moreover, diet‐driven differences in the inhibitory activity of the hemolymph were tested against Escherichia coli DH5α and Micrococcus yunnanensis HI55 using diffusion assays in solid media. Results showed that a municipal organic waste‐based diet produced a significant overexpression of antimicrobial peptides only in prepupae. Inclusion of vegetable oils caused an upregulation of at least one peptide, except for the corn oil. Higher expression of both genes was observed when sunflower oil was added 5 days before pupation. All hemolymph samples showed an inhibitory activity against bacteria colonies. Our results suggest that municipal organic waste‐based diet and vegetable oil‐added diet may successfully impact the immune system of H. illucens. Such alternatives may also exist for other species of economic interest. [ABSTRACT FROM AUTHOR]

The recent progressive increase in the incidence of invasive fungal infections, especially in immunocompromised patients, makes the search for new therapies crucial in the face of the growing drug resistance of prevalent nosocomial yeast strains. The latest research focuses on the active compounds of natural origin, inhibiting fungal growth, and preventing the formation of fungal biofilms. Antimicrobial peptides are currently the subject of numerous studies concerning effective antifungal therapy. In the present study, the antifungal properties of two synthetic peptides (ΔM3, ΔM4) derived from an insect antimicrobial peptide -- cecropin D -- were investigated. The fungicidal activity of both compounds was demonstrated against the yeast forms of Candida albicans, Candida tropicalis, and Candida parapsilosis, reaching a MFC99.9 in the micromolar range, while Candida glabrata showed greater resistance to these peptides. The scanning electron microscopy revealed a destabilization of the yeast cell walls upon treatment with both peptides; however, their effectiveness was strongly modified by the presence of salt or plasma in the yeast environment. The transition of C. albicans cells from yeast to filamentous form, as well as the formation of biofilms, was effectively reduced by ΔM4. Mature biofilm viability was inhibited by a higher concentration of this peptide and was accompanied by increased ROS production, activation of the GPX3 and SOD5 genes, and finally, increased membrane permeability. Furthermore, both peptides showed a synergistic effect with caspofungin in inhibiting the metabolic activity of C. albicans cells, and an additive effect was also observed for the mixtures of peptides with amphotericin B. The results indicate the possible potential of the tested peptides in the prevention and treatment of candidiasis. [ABSTRACT FROM AUTHOR]

The black soldier fly (BSF), Hermetia illucens L. (Diptera: Stratiomyidae), has a great bioconversion potential and ability to develop on diverse substrates. Although the use of catering waste and food by-products containing meat and fish would reduce the footprint of the insect sector, to date, in Europe, their use is still facing legal obstacles for insects as food and feed. Since a major request from the EU insect sector is to diversify the spectrum of allowed substrates, and considering that variations in diet composition could influence insect immune responses, we evaluated the impact of different catering wastes on BSF prepupae immunity. Insects were reared on five diets: one based on feed for laying hens and four based on catering waste containing (i) fruits and vegetables; (ii) fruits, vegetables and bread; (iii) fruit, vegetables, bread and dairy products; (iv) fruits, vegetables, bread, meat and fish. The gene expression of two antimicrobial peptides (AMPs), one defensin and one cecropin, was assessed. Moreover, the hemolymph inhibitory activity against Escherichia coli DH5α and Micrococcus yunnanensis HI55 was evaluated using diffusion assays in solid media. The up-regulation of both AMPs' encoding genes was observed in insects fed a bread-added and dairy product-added diet. All hemolymph samples showed inhibitory activity against both bacteria, affecting the colony size and number. The obtained results show how catering waste positively influences the H. illucens immune system. The possibility of modulating AMP expression levels through the diet opens up new perspectives in the management of insect health in mass rearings. [ABSTRACT FROM AUTHOR]

The recent progressive increase in the incidence of invasive fungal infections, especially in immunocompromised patients, makes the search for new therapies crucial in the face of the growing drug resistance of prevalent nosocomial yeast strains. The latest research focuses on the active compounds of natural origin, inhibiting fungal growth, and preventing the formation of fungal biofilms. Antimicrobial peptides are currently the subject of numerous studies concerning effective antifungal therapy. In the present study, the antifungal properties of two synthetic peptides (ΔM3, ΔM4) derived from an insect antimicrobial peptide – cecropin D – were investigated. The fungicidal activity of both compounds was demonstrated against the yeast forms of Candida albicans, Candida tropicalis, and Candida parapsilosis, reaching a MFC99.9 in the micromolar range, while Candida glabrata showed greater resistance to these peptides. The scanning electron microscopy revealed a destabilization of the yeast cell walls upon treatment with both peptides; however, their effectiveness was strongly modified by the presence of salt or plasma in the yeast environment. The transition of C. albicans cells from yeast to filamentous form, as well as the formation of biofilms, was effectively reduced by ΔM4. Mature biofilm viability was inhibited by a higher concentration of this peptide and was accompanied by increased ROS production, activation of the GPX3 and SOD5 genes, and finally, increased membrane permeability. Furthermore, both peptides showed a synergistic effect with caspofungin in inhibiting the metabolic activity of C. albicans cells, and an additive effect was also observed for the mixtures of peptides with amphotericin B. The results indicate the possible potential of the tested peptides in the prevention and treatment of candidiasis.

Abstract Background The recent emergence of antibiotic-resistant strains of bacteria has increased the need to develop effective alternatives to antibiotics. Antimicrobial peptides have been considered as a promising product with several advantages. Results In this present study, we identified a novel cecropin from the armyworm, Mythimna separata (armyworm cecropin 1, AC-1) by transcriptome sequencing and multi-sequence alignment analysis. The AC-1 precursor comprised 63 amino acid residues, containing a conserved cleavage site of the signal peptide, Ala23-Pro24, while the mature AC-1 included 39 amino acid residues. Chemically synthesized AC-1 exhibited low hemolytic activity against chicken red blood cells, low cytotoxicity against swine testis cells, and effective antimicrobial activity against Salmonella, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa. Its antimicrobial activity against Salmonella remained after incubation for 1 h at 100 °C or in 250 mM NaCl, KCl, or MgCl2 solution, implying good thermal- and salt-resistant stabilities. The bactericidal effect of AC-1 on E. coli gradually increased with increasing AC-1 concentration, resulting in deformation, severe edema, cytolysis, cell membrane damage, and reducing intracellular electron density. Additionally, recombinant AC-1 protein expressed in E. coli was digested by enterokinase protease to obtain AC-1, which showed similar antimicrobial activity against E. coli to chemically synthesized AC-1. Conclusions This study identified a novel antimicrobial peptide that may represent a potential alternative to antibiotics.

Following a ban on antibiotic use in the feed industry, trials on the effects of various immunostimulants (prebiotics, probiotics, antimicrobial peptides [AMPs], and herbs) on the survival, growth, immunity, and disease control of farmed fish in aquaculture are being rapidly conducted. The wide variety of microbes with roles in nutrition, metabolism, and immunity in the fish intestine is the primary factor affecting the fermentability and functionality of dietary immunostimulants. For this reason, the dynamic interactions between immunostimulants and the intestinal microbiome may influence fish health. In this study, the effects of two agriculturally important AMPs (nisin and cecropin) and one herb (Penthorum chinense) on the gut microbiome of common carp were investigated, using 16S rDNA high-throughput sequencing. The results suggest that all three substances can alter the richness, diversity, and composition of the intestinal microbiota of common carp. P. chinense had a similar effect on the gut microbiota of common carp to that of nisin, and both promoted more striking changes in the gut microbiota community than did cecropin. The relative abundance of Proteobacteria was lower in the nisin and P. chinense groups than in the control and cecropin groups. The relative abundance of Bacteroidetes in the nisin, cecropin, and P. chinense groups was markedly increased, compared with that of the control group. Additionally, nisin, cecropin, and P. chinense showed obvious anti-inflammatory effects on the fish intestine, which was reflected by significantly increasing the expression levels of two anti-inflammatory cytokines IL-10 and TGF-β. Some digestive enzyme activities in the fish intestine were also significantly enhanced by supplementing these three substances in feeds.

Abstract Background It has been reported that there are more than ten antimicrobial peptides (AMPs) belonging to the cecropin family in Musca domestica; however, few of them have been identified, and the functions of the other molecules are poorly understood. Methods Sequences of the M. domestica cecropin family of genes were cloned from cDNA template, which was reverse-transcribed from total mRNA isolated from third-instar larvae of M. domestica that were challenged with pathogens. Sequence analysis was performed using DNAMAN comprehensive analysis software, and a molecular phylogenetic tree of the cecropin family was constructed using the Neighbour-Joining method in MEGA v.5.0 according to the mature peptide sequences. Antibacterial activity of the synthetic M. domestica cecropin protein was detected and the minimum inhibitory concentration (MIC) values were determined using broth microdilution techniques. Time-killing assays were performed on the Gram-negative bacteria, Acinetobacter baumannii, at the logarithmic or stabilizing stages of growth, and its morphological changes when treated with Cec4 were assessed by scanning electron microscopy (SEM) and detection of leakage of 260 nm absorbing material. Results Eleven cecropin family genes, namely Cec01, Cec02 and Cec1-9, show homology to the Cec form in a multigene family on the Scaffold18749 of M. domestica. In comparing the encoded cecropin protein sequences, most of them have the basic characteristics of the cecropin family, containing 19 conservative amino acid residues. To our knowledge, this is the first experimental demonstration that most genes in the Cec family are functional. Cec02, Cec1, Cec2, Cec5 and Cec7 have similar antibacterial spectra and antibacterial effects against Gram-negative bacteria, while Cec4 displays a more broad-spectrum of antimicrobial activity and has a very strong effect on A. baumannii. Cec4 eliminated A. baumannii in a rapid and concentration-dependent manner, with antibacterial effects within 24 h at 1× MIC and 2× MIC. Furthermore, SEM analysis and the leakage of 260 nm absorbing material detection indicated that Cec4 sterilized the bacteria through the disruption of cell membrane integrity. Conclusions Although there are more than ten cecropin genes related to M. domestica, some of them have no preferred antibacterial activity other than Cec4 against A. baumannii.

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Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans. Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans. Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides. [ABSTRACT FROM AUTHOR]

The oral microbiome has major impacts on oral health and disease. Antimicrobial peptides (AMPs), such as nisin and cecropin, have been widely used as food preservatives or feed additives, and are thus inevitably ingested by consumers through their oral cavity. However, as broad-spectrum antimicrobial reagents, the effect of AMPs on the oral microbiome of consumer’s remains poorly characterized. In this study, we performed 16S rDNA high-throughput sequencing to investigate the effect of nisin and cecropin on the oral microbiomes of rats. Our results suggest that although nisin and cecropin have different effects on the oral microbiome of rats, both AMPs impact the composition of oral microbial communities at the phylum and genus levels. Cecropin significantly reduced the diversity and richness of rat oral microbial communities. Notably, the relative abundance of the pathogen Acinetobacter baumannii increased in the oral microbial community of rats fed cecropin-containing feed. In addition, nisin significantly reduced the amount of secretory immunoglobulin A in the saliva of rats.

Abstract Background Mosquitoes are armed with physiologically active compounds to suppress the host immunity including host inflammatory reaction. However, the specific anti-inflammatory components in mosquitoes remain unknown. Results By searching for the immunomodulatory molecules from the mosquito Aedes aegypti (Diptera: Culicidae) at NCBI for anti-inflammatory function, five cecropins (for short in this study: AeaeCec1, 2, 3, 4 and 5) were selected. AeaeCec1-5 efficiently inhibited the expression of inducible nitric oxide synthase (iNOS), nitrite, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human peripheral blood mononuclear cells (PBMCs) with low toxicity to mammalian cells. Among the five analogues, AeaeCec5 had the strongest anti-inflammatory activity, and generated an additive effect with other AeaeCec peptides. In a mouse model of endotoxin shock, AeaeCec1-5 effectively reduced TNF-α, IL-1β and IL-6 expression in lungs, serum and peritoneal lavage and correspondingly reduced lung damage and edema, with AeaeCec5 showing the best protection. In mice infected with Escherichia coli or Pseudomonas aeruginosa, administration of AeaeCec5 reduced the production of TNF-α, IL-1β and IL-6 and correspondingly reduced lung tissue damage. These effects of Ae. aegypti AeaeCec1-5 were attributed to an efficient inhibition of the activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signaling pathways, as well as partial neutralization of LPS. Conclusions The current work characterized the specific anti-inflammatory agents in Ae. aegypti and provided AeaeCec5 as a potent anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory therapy.

Antimicrobial peptides (AMPs) are considered to be candidate effectors for eliciting humoral immune responses against infectious pathogens in the host. Cecropins are α‐helical peptides of 30–40 amino acids, which are known to permeabilize bacterial membranes, and play authoritative roles in the innate immune system of insects. In the present study, we identified the full‐length open reading frame (ORF) encoding the Tenebrio molitor cecropin‐2 (TmCec2) gene using the Tribolium castaneum cecropin‐2 (TcCec2) gene to query a T. molitor Rnaseq database. Phylogenetic analysis revealed close identity of TmCec2 with TcCec2. TmCec2 was ubiquitously expressed in the insect during all developmental stages, with the highest expression observed in the adult. Tissue‐specific TmCec2 expression was highest in larval hemocytes and in the adult integument and hemocytes. Microbial challenge experiments revealed that TmCec2 was highly induced in response to gram‐positive and gram‐negative bacteria, and fungi. These data provide credible evidence for a putative role of TmCec2 in insect innate immunity against a plethora of pathogens. [ABSTRACT FROM AUTHOR]

Chronic exposure to UVB radiation causes photoaging, immunosuppression, and ultimately photocarcinogenisis through the generation of reactive oxygen species (ROS). The ability of natural compounds in neutralizing the effects of oxidative stress is being explored with increased interest. Silk sericin, a biopolymer is reported to have diverse biological properties. In an effort to make the silk sericin pure, more effective and multifunctional, we have recombinantly expressed both functional sericin as well as sericin-cecropin B fusion proteins. Herein, we studied the antioxidant and anti-UVB potential of recombinant sericin and sericin-cecropin B proteins against oxidative stress using human primary dermal fibroblast cells. Treating the cells with recombinant sericin (RS) or sericin-cecropin B (RSC) prior to exposure to UVB and H2O2, effectively increased the cell viability by approximately 30% and 50%, respectively, in comparison to non-treated control. The protective effects were further evident in terms of significant reduction of LDH in oxidatively challenged cells treated with RS and RSC. A reduction in LDH release of at least 16 and 33% was observed with RS and RSC treatments, respectively, in comparison to exposed control. Further, elevated levels of catalase and superoxide dismutase (SOD) activity were observed. Importantly, the RSC fusion protein exhibited enhanced protective effects than cells treated with RS alone. Our results demonstrate that the functional attributes of cecropin B along with sericin activity in the fusion protein conferred enhanced protection against UVB- and H2O2-induced oxidative damage in human dermal fibroblasts. The improved antioxidant activity of recombinant sericin fusion biopolymer has great potential as a promising therapeutic agent for ROS-induced skin diseases. [ABSTRACT FROM AUTHOR]

Background: The recent emergence of antibiotic-resistant strains of bacteria has increased the need to develop effective alternatives to antibiotics. Antimicrobial peptides have been considered as a promising product with several advantages. Results: In this present study, we identified a novel cecropin from the armyworm, Mythimna separata (armyworm cecropin 1, AC-1) by transcriptome sequencing and multi-sequence alignment analysis. The AC-1 precursor comprised 63 amino acid residues, containing a conserved cleavage site of the signal peptide, Ala23-Pro24, while the mature AC-1 included 39 amino acid residues. Chemically synthesized AC-1 exhibited low hemolytic activity against chicken red blood cells, low cytotoxicity against swine testis cells, and effective antimicrobial activity against Salmonella, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa. Its antimicrobial activity against Salmonella remained after incubation for 1 h at 100 °C or in 250 mM NaCl, KCl, or MgCl2 solution, implying good thermal- and salt-resistant stabilities. The bactericidal effect of AC-1 on E. coli gradually increased with increasing AC-1 concentration, resulting in deformation, severe edema, cytolysis, cell membrane damage, and reducing intracellular electron density. Additionally, recombinant AC-1 protein expressed in E. coli was digested by enterokinase protease to obtain AC-1, which showed similar antimicrobial activity against E. coli to chemically synthesized AC-1. Conclusions: This study identified a novel antimicrobial peptide that may represent a potential alternative to antibiotics. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides are potential alternatives to traditional antibiotics in the face of increasing bacterial resistance. Insects possess many antimicrobial peptides and have become a valuable source of novel and highly effective antimicrobial peptides. Hermetia illucens as a resource insect, for example, has the highest number of antimicrobial peptides of any dipteran. However, most antimicrobial peptides, especially cecropin, have not been comprehensively identified and have not been evaluated for their antimicrobial ability. In this study, we analyzed the localization and gene structure of 33 cecropin molecules in the H. illucens genome and evaluated their activity against common human pathogens. The results showed that 32 cecropin molecules were concentrated on 1 chromosome, most with 2 exons. More importantly, most of the cecropins had a good antibacterial effect against Gram-negative bacteria, and were not hemolytic. The minimum inhibitory concentration (MIC) of the cecropin designated H3 against E. coli was 4 μg/mL. The toxicity, killing time kinetics, and anti-biofilm activity of H3 were further investigated and confirmed its antimicrobial ability. Overall, H3 is a potential candidate for the development of new antimicrobials to treat severe infections caused by Gram-negative pathogens such as E. coli. [Display omitted] • The genome of H. illucens contains 33 cecropin molecules, most of which are located on one chromosome. • There are several cecropin molecules with tandem repeats that encode the same mature peptide. • Of the 15 mature cecropin peptides, cecropin H3 was particularly active against E. coli. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9-mediated m ultiplex g enome e diting (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel. In this study, we used s ingle- s gRNA-based g enome e diting (ssGE) and m ulti- s gRNA-based MGE (msMGE) to replace the luteinizing hormone (lh) and melanocortin-4 receptor (mc4r) genes with the cathelicidin (As - Cath) transgene and the myostatin (two target sites: mstn1 , mstn2) gene with the cecropin (Cec) transgene, respectively. A total of 9000 embryos were microinjected from three families, and 1004 live fingerlings were generated and analyzed. There was no significant difference in hatchability (all P > 0.05) and fry survival (all P > 0.05) between ssGE and msMGE. Compared to ssGE, CRISPR/Cas9-mediated msMGE assisted by the mixture of dsDNA and dcPlasmid donors yielded a higher knock-in (KI) efficiency of As-Cath (19.93 %, [59/296] vs. 12.96 %, [45/347]; P = 0.018) and Cec (22.97 %, [68/296] vs. 10.80 %, [39/361]; P = 0.003) transgenes, respectively. The msMGE strategy can be used to generate transgenic fish carrying two transgenes at multiple loci. In addition, double and quadruple mutant individuals can be produced with high efficiency (36.3 % ∼ 71.1 %) in one-step microinjection. In conclusion, we demonstrated that the CRISPR/Cas9-mediated msMGE allows the one-step generation of simultaneous insertion of the As-Cath and Cec transgenes at four sites, and the simultaneous disruption of the lh , mc4r , mstn1 and mstn2 alleles. This msMGE system, aided by the mixture donors, promises to pioneer a new dimension in the drive and selection of multiple designated traits in other non-model organisms. [ABSTRACT FROM AUTHOR]

The increasing antimicrobial-resistant prevalence has become a severe health problem. It has led to the invention of a new antimicrobial agent such as antimicrobial peptides. Heteroscorpine-1 is an antimicrobial peptide that has the ability to kill many bacterial strains. It consists of 76 amino acid residues with a cecropin-like region in N-terminal and a defensin-like region in the C-terminal. The cecropin-like region from heteroscorpine-1 (CeHS-1) is similar to cecropin B, but it lost its glycine-proline hinge region. The bioinformatics prediction was used to help the designing of mutant peptides. The addition of glycine-proline hinge and positively charged amino acids, the deletion of negatively charged amino acids, and the optimization of the hydrophobicity of the peptide resulted in two mutant peptides, namely, CeHS-1 GP and CeHS-1 GPK. The new mutant peptide showed higher antimicrobial activity than the native peptide without increasing toxicity. The interaction of the peptides with the membrane showed that the peptides were capable of disrupting both the inner and outer bacterial cell membrane. Furthermore, the SEM analysis showed that the peptides created the pore in the bacterial cell membrane resulted in cell membrane disruption. In conclusion, the mutants of CeHS-1 had the potential to develop as novel antimicrobial peptides.

BACKGROUND: Antimicrobial peptides, an extract from nature, are a basic component of host immunity that make toxic effect on highly proliferative cells. They have attracted extensive attention of scientists. The understanding of the antineoplastic mechanism of antimicrobial peptides can contribute to its application in clinical practice. OBJECTIVE: To summarize the research advances in antineoplastic mechanism of antimicrobial peptides. METHODS: The first author conducted a computer-based retrieval of PubMed, Springerlink, Web of Science, and ScienceDirect databases for relevant articles published from January 2015 to May 2019. The keywords were "antimicrobial, peptide, antitumor mechanisms, antitumor activity and anti-neoplastic". The articles concerning antineoplastic mechanism of antimicrobial peptides and research progress were selected. RESULTS AND CONCLUSION: Cationic antimicrobial peptides synthesized by ribosomes and the host defense peptides can interact with the membrane of bacteria, which showed a broad-spectrum antimicrobial activity. Compared with normal cells, the proportion of phosphatidylserine on the surface of cancer cells, which is negatively charged, is increased dramatically. As a result, the cationic amphiphilic peptides are good candidate for the antineoplastic drugs, and possess a high selectivity. There are two major antitumor mechanism of antimicrobial peptides, which are selective membrane destruction and non-membrane dissolution (α-defensin-1 and lactoferrin B). The clinical application of antimicrobial peptides against tumors is mainly restricted by their stability and the ways to administration. By optimizing its structure and drug delivery systems, these antimicrobial peptides will play a critical role in the cancer treatment. [ABSTRACT FROM AUTHOR]

Background: It has been reported that there are more than ten antimicrobial peptides (AMPs) belonging to the cecropin family in Musca domestica; however, few of them have been identified, and the functions of the other molecules are poorly understood. Methods: Sequences of the M. domestica cecropin family of genes were cloned from cDNA template, which was reverse-transcribed from total mRNA isolated from third-instar larvae of M. domestica that were challenged with pathogens. Sequence analysis was performed using DNAMAN comprehensive analysis software, and a molecular phylogenetic tree of the cecropin family was constructed using the Neighbour-Joining method in MEGA v.5.0 according to the mature peptide sequences. Antibacterial activity of the synthetic M. domestica cecropin protein was detected and the minimum inhibitory concentration (MIC) values were determined using broth microdilution techniques. Time-killing assays were performed on the Gram-negative bacteria, Acinetobacter baumannii, at the logarithmic or stabilizing stages of growth, and its morphological changes when treated with Cec4 were assessed by scanning electron microscopy (SEM) and detection of leakage of 260 nm absorbing material. Results: Eleven cecropin family genes, namely Cec01, Cec02 and Cec1-9, show homology to the Cec form in a multigene family on the Scaffold18749 of M. domestica. In comparing the encoded cecropin protein sequences, most of them have the basic characteristics of the cecropin family, containing 19 conservative amino acid residues. To our knowledge, this is the first experimental demonstration that most genes in the Cec family are functional. Cec02, Cec1, Cec2, Cec5 and Cec7 have similar antibacterial spectra and antibacterial effects against Gram-negative bacteria, while Cec4 displays a more broad-spectrum of antimicrobial activity and has a very strong effect on A. baumannii. Cec4 eliminated A. baumannii in a rapid and concentration-dependent manner, with antibacterial effects within 24 h at 1× MIC and 2× MIC. Furthermore, SEM analysis and the leakage of 260 nm absorbing material detection indicated that Cec4 sterilized the bacteria through the disruption of cell membrane integrity. Conclusions: Although there are more than ten cecropin genes related to M. domestica, some of them have no preferred antibacterial activity other than Cec4 against A. baumannii. [ABSTRACT FROM AUTHOR]

• Two isolates of Metarhizium robersii produce microsclerotia similarly. • Microsclerotia from Metarhizium robersii are pathogenic against Aedes aegypti larvae. • The fungus-larva interaction results on a delayed host response assisting mortality. Aedes aegypti is a vector of various disease-causing arboviruses. Chemical insecticide-based methods for mosquito control have increased resistance in different parts of the world. Thus, alternative control agents such as the entomopathogenic fungi are excellent candidates to control mosquitoes as part of an ecofriendly strategy. There is evidence of the potential of entomopathogenic fungal conidia and blastospores for biological control of eggs, larval and adult stages, as well as the pathogenicity of fungal microsclerotia against adults and eggs. However, there are no studies on the pathogenicity of microsclerotia against either aquatic insects or insects that develop part of their life cycle in the water, such as the A. aegypti larvae. In this study, we assayed the production of microsclerotia and their pathogenicity against A. aegypti larvae of two isolates of Metarhizium robertsii , i.e., CEP 423 isolated in La Plata, Argentina, and the model ARSEF 2575. Both isolates significantly reduced the survival of A. aegypti exposed to their microsclerotia. The fungus-larva interaction resulted in a delayed response in the host. This was evidenced by the expression of some humoral immune system genes such as defensins and cecropin on the 9th day post-infection, when the fungal infection was consolidated as a successful process that culminates in larvae mortality. In conclusion, M. robertsii microsclerotia are promising propagules to be applied as biological control agents against mosquitoes since they produce pathogenic conidia against A. aegypti larvae. [Display omitted] [ABSTRACT FROM AUTHOR]

Increasing microbial resistance, coupled with a lack of new antimicrobial discovery, has led researchers to refocus on antimicrobial peptides (AMPs) as novel therapeutic candidates. Significantly, the less toxic cecropins have gained widespread attention for potential antibacterial agent development. However, the narrow activity spectrum and long sequence remain the primary limitations of this approach. In this study, we truncated and modified cecropin 4 (41 amino acids) by varying the charge and hydrophobicity balance to obtain smaller AMPs. The derivative peptide C18 (16 amino acids) demonstrated high antibacterial activity against Gram-negative and Gram-positive bacteria, as well as yeasts. Moreover, C18 demonstrated a minimal inhibitory concentration (MIC) of 4 µg/mL against the methicillin-resistant Staphylococcus aureus (MRSA) and showed synergy with daptomycin with a fractional inhibition concentration index (FICI) value of 0.313. Similar to traditional cecropins, C18 altered the membrane potential, increased fluidity, and caused membrane breakage at 32 µg/mL. Importantly, C18 eliminated 99% persisters at 10 × MIC within 20 min and reduced the biofilm adherence by ~40% and 35% at 32 and 16 µg/mL. Besides, C18 possessed a strong binding ability with DNA at 7.8 μM and down-regulated the expression of virulence factor genes like agrA, fnb-A, and clf-1 by more than 5-fold (p < 0.05). Interestingly, in the Galleria mellonella model, C18 rescued more than 80% of larva infected with the MRSA throughout 120-h post-infection at a single dose of 8 mg/kg (p < 0.05). In conclusion, this study provides a reference for the transformation of cecropin to derive small peptides and presents C18 as an attractive therapeutic candidate to be developed to treat severe MRSA infections.

Abstract Background A diverse group of physiologically active peptides/proteins are present in the salivary glands of horsefly Tabanus yao (Diptera, Tabanidae) that facilitate acquisition of blood meal. However, their roles in the regulation of local inflammation remains poorly understood. Methods Induction expression profiles of immune-related molecules in the salivary glands of T. yao was analyzed by quantitative PCR (qPCR) after bacterial feeding. A significantly up-regulated molecule (cecropin-TY1) was selected for anti-inflammatory assay in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. The transcription levels of inducible NO synthase (iNOS) and pro-inflammatory cytokines were quantified by qPCR. Nitric oxide (NO) production was determined by Griess reagent. Pro-inflammatory cytokine production was determined by an enzyme-linked immunosorbent assay (ELISA). The inflammatory signals were assayed by Western blotting analysis. The secondary structure of cecropin-TY1 was measured by Circular dichroism (CD) spectroscopy. Interaction of cecropin-TY1 with LPS was evaluated by the dissociation of fluorescein isothiocyanate (FITC)-conjugated LPS aggregates and neutralization of LPS determined by a quantitative Chromogenic End-point Tachypleus amebocyte lysate (TAL) assay kit. Homology modeled structure analysis and mutation of key residues/structures were performed to understand its structure-activity relationship. Results Cecropin-TY1 was demonstrated to possess high anti-inflammatory activity and low cytotoxicity toward mouse macrophages. In LPS-stimulated mouse peritoneal macrophage, addition of cecropin-TY1 significantly inhibited the production of nitric oxide (NO) and pro-inflammatory cytokines. Further study revealed that cecropin-TY1 inhibited inflammatory cytokine production by blocking activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signals. Cecropin-TY1 even interacted with LPS and neutralized LPS. The secondary structure analysis revealed that cecropin-TY1 adopted unordered structures in hydrophobic environment but converted to α-helical confirmation in membrane mimetic environments. Homology modeled structure analysis demonstrated that cecropin-TY1 adopted two α-helices (Leu3-Thr24, Ile27-Leu38) linked by a hinge (Leu25-Pro26) and the structure surface was partly positively charged. Structure-activity relationship analysis indicated that several key residues/structures are crucial for its anti-inflammatory activity including α-helices, aromatic residue Trp2, positively charged residues Lys and Arg, hinge residue Pro26 and N-terminal amidation. Conclusions We found a novel anti-inflammatory function of horsefly-derived cecropin-TY1 peptide, laying groundwork for better understanding the ectoparasite-host interaction of horsefly with host and highlighting its potency in anti-inflammatory therapy for sepsis and endotoxin shock caused by Gram-negative bacterial infections.

Abstract Background Several antimicrobial peptides (AMPs) belonging to the cecropin family have been identified from the salivary glands of different black fly species, however, the immunological functions for these molecules were poorly understood. Methods A novel cecropin-like antimicrobial peptide (SibaCec) was purified using reverse phase high-performance liquid chromatography (RP-HPLC) from the salivary glands of the black fly Simulium bannaense. The amino acid sequence of SibaCec was determined by a combination method of automated Edman degradation and cDNA sequencing. The morphologic changes of Gram-negative bacteria Escherichia coli treated with SibaCec were assessed by scanning electron microscopy (SEM). Quantitative PCR (qPCR) was performed to analyze the mRNA expression of the inducible NO synthase (iNOS) and pro-inflammatory cytokines. Nitric oxide (NO) generation was examined using a Griess assay and the secretion of pro-inflammatory cytokines was determined by an enzyme-linked immunosorbent assay (ELISA). The activation of extracellular signal-regulated kinase (ERK), p38, and the nuclear translocation of nuclear factor-kappaB (NF-κB) were assessed by Western blotting analysis. Circular dichroism (CD) spectroscopy was performed to evaluate the secondary structure of SibaCec in solvent environment. Interaction of SibaCec with lipopolysaccharide (LPS) was studied using fluorescein isothiocyanate (FITC)- conjugated LPS aggregates. Neutralization of LPS by SibaCec was assayed with the chromogenic limulus amebocyte lysate (LAL) test. qPCR was also used to analyze the expression of SibaCec mRNA in the salivary glands of insects after oral infection with the bacteria E.coli. Results SibaCec possessed potent antimicrobial activity against Gram-negative bacteria, and showed low cytotoxicity toward mammalian cells. SEM analysis indicated that SibaCec killed bacteria through the disruption of cell membrane integrity. Furthermore, SibaCec significantly inhibited lipopolysaccharide (LPS)-induced production of NO and pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interferon-1β (IL-1β) and interferon-6 (IL-6) by blocking the activation of MAPKs and NF-κB signaling pathways. It mainly adopted an α-helix conformation in membrane-mimetic environments. SibaCec could interact and neutralize LPS. Infection of black flies with bacteria caused an upregulation of the expression of SibaCec. Conclusions These results demonstrated that in addition to the bactericidal capacity, SibaCec can function as immune regulator, inhibiting host secretion of inflammatory factors.

Cecropins (Cec) are antibacterial peptides and their expression is induced in a pig intestinal parasite Ascaris suum by bacterial infection. To explore the usefulness of its activity as an antibiotic, CecP4 cDNA was prepared and cloned into the pPICZ B expression vector and followed by the integration into AOX1 locus in Pichia pastoris. The supernatants from cell culture were collected after methanol induction and concentrated for the test of antimicrobial activity. The recombinant P. patoris having CecP4 showed antimicrobial activity when tested against Staphyllococcus aureus in disc diffusion assay. We selected one of the CecP4 clones (CecP4-2) and performed further studies with it. The growth of recombinant P. pastoris was optimized using various concentration of methanol, and it was found that 2% methanol in the culture induced more antibacterial activity, compared to 1% methanol. We extended the test of antimicrobial activity by applying the concentrated supernatant of CecP4 culture to Pseudomonas aeruginosa and E. coli respectively. Recombinant CecP4 also showed antimicrobial activity against both Pseudomona and E. coli, suggesting the broad spectrum of its antimicrobial activity. After improvements for the scale-up, it will be feasible to use recombinant CecP4 for supplementation to the feed to control microbial infections in young animals, such as piglets.

Cecropin A-Magainin 2 (CA-MA) hybrid antimicrobial peptide (AMP), a combination of two naturally occurring AMPs, cecropin A and magainin 2 is preferred widely in biotechnological, nano and pharmaceutical applications. It exhibits a strong antibacterial activity with a characteristic reduced cytotoxic effect towards mammalian cells. In this study, three AMP structures native CA-MA hybrid and its tryptophan substitutes CA-MA L2 and CA-MA A2 was computationally studied to analyze their structural stability and functionality. Computational analysis like, intra-molecular interactions (25), relative stability (3.22) and instability index (−14.28) showed an increase in structural stability of native CA-MA hybrid. Additionally, the generated peptide ensembles showed a RMSD (3.98 Å), RMSF (0.202 Å), radius of gyration (11.98 Å), ovality (3.33) and hydrophobicity (69.7%) supporting native CA-MA along with hydrogen bond strength (−4.212 kcal/mol) and distribution comparatively. The distribution of secondary structure in native CA-MA hybrid showed the sequential maintenance of stable helical content along with helical stability (52.25%) and computed free energy (−1.74 kcal/mol) in membrane mimicking environment proving its functional activity comparatively. This study aids in designing stable AMP biodrugs with low cytotoxicity in future, the result can be potentially extended to other AMPs to assist in their exploitation as peptide and nano drugs. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the anti-inflammatory activity of Musca domestica cecropin-A2 (Mdc-A2) toward Staphylococcus aureus ( S. aureus ) to learn more about their immunological functions. RAW264.7 cells were transfected with recombinant lentiviruses introduce pLEX-Mdc-A2into the RAW264.7 cell line (RAW-Mdc-A2). The RAW264.7 cell line with empty pLEX (RAW-pLEX) was produced in the same manner as a negative control. Real-time quantitative reverse transcription PCR (RT-PCR) was performed to analyze the mRNA expression of TNF-a, IL-1β, NFκB-1 and NFκB-2 in S. aureus -stimulated RAW-Mdc-A2 cells and RAW-pLEX cells in untreated cells and cells treated for 3 h, 6 h, 12 h and 24 h. RT-PCR was performed to analyze the mRNA expression of TNF-a, NFκB-1 and NFκB-2 stimulated by Lipoteichoic acid (LTA). Production of TNF-a was detected by enzyme-linked immunosorbent assay (ELISA). Colony counts were used to calculate the number of CFU per mL of cell culture supernatants. The results showed that compared to RAW-pLEX cells, stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of TNF-a transcript variant 1 (TNF-a-tv-1) at 6 h and 12 h and the mRNA expression of TNF-a transcript variant 2 (TNF-a-tv-2) at 3 h, 6 h and 12 h. Compared to RAW-pLEX cells, stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of IL-1β-T at 3 h, 6 h and 12 h as well as the mRNA expression of IL-1β at 3 h and 6 h. The expression and production of TNF-a and bacterial burden of cell culture supernatants were significantly down-regulated in RAW-Mdc-A2 cells stimulated by S. aureus , and the expression and production of TNF-a were significantly down-regulated in RAW-Mdc-A2 cells stimulated by LTA. Compared to RAW-pLEX cells, stable transfection of Mdc-A2 in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of NFκB-1 at 3 h, 6 h and 12 h as well as the mRNA expression of NFκB-2 at 6 h. Additionally, stable transfection of Mdc-A2 in RAW264.7 cells stimulated by LTA significantly down-regulated the mRNA expression of NFκB-1. In conclusion, Mdc-A2 possesses potent anti-inflammatory activity and potent antimicrobial activity. Additionally, Mdc-A2 may interact with LTA and execute strong anti-inflammatory activity by blocking the activation of NF-κB signaling pathways. [ABSTRACT FROM AUTHOR]

The insect immune system has versatile ways of coping with microbial insults. Currently, innate immune priming has been described in several invertebrates, and the first insights into its mechanistic basis have been described. Here we studied infections with two different strains of Serratia marcescens bacteria in two different Lepidopteran hosts. The results reveal fundamental differences between the two hosts, a well-known model organism Galleria mellonella and a non-model species Arctia plantaginis. They differ in their strategies for resisting oral infections; priming their defences against a recurring sepsis; and upregulating immunity related genes as a response to the specific pathogen strains. The two bacterial strains (an environmental isolate and an entomopathogenic isolate) differ in their virulence, use of extracellular proteases, survival in the larval gut, and in the immune response they evoke in the hosts. This study explores the potential mechanistic explanations for both host and pathogen specific characters that significantly affect the outcome of Gram-negative bacterial infection in Lepidopteran larvae. The results highlight the need to pay greater attention to the differences between model and non-model hosts, and closely related pathogen strains, in immunological studies. [ABSTRACT FROM AUTHOR]

Cecropins are basic antibacterial peptides that have potent antimicrobial activities. We induced and purified a novel antimicrobial peptide exhibiting activity against Gram-negative bacteria from the immunized hemolymph of Hermetia illucens larvae . The immunized hemolymph was extracted, and the novel cecropin-like peptide 1 (CLP1) was purified using solid-phase extraction and reverse-phase chromatography. The purified CLP1 demonstrated a molecular weight of 4,840 Da, as determined by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF). From analysis of CLP1 by N-terminal amino acid sequencing using Edman degradation, combined with MALDI-TOF and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), the amino acid sequence of the mature peptide was determined to be GWRKRVFKPVEKFGQRVRDAGVQGIAIAQQGANVLATARGGP PQQG. In NCBI BLAST, the amino acid sequence of CLP1 was found to be 60 % identical to the Drosophila melanogaster cecropin C. In silico analysis revealed that CLP1 was suggested to be part of the cecropin superfamily of AMPs characterized as cationic, linear, α-helical, and amphipathic polypeptides. Analysis of the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) showed that CLP1 exerted antibacterial effects against Gram-negative bacteria. The expression of CLP1 transcripts in several tissues after bacterial challenge was measured by quantitative real-time PCR. CLP1 expression was negligible throughout the body before immunization, and was mostly evident in the fat body after immunization. [ABSTRACT FROM AUTHOR]