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Background: Cecropin P1, acting as an antimicrobial, has a broad-spectrum antibacterial activity with some antiviral and antifungal properties. It is a promising natural alternative to antibiotics which is originally isolated from the pig intestinal parasitic nematode Ascaris suum. Many studies have shown that Cecropin P1 is helpful for the prevention or treatment of clinical diseases. Therefore, it is very necessary to establish a safe, nontoxic, and efficient expression method of Cecropin P1. Results: The results indicated that the recombinant protein was about 5.5 kDa showed by Tricine–SDS–PAGE and Western blot. And Cecropin P1 was efficiently secreted and expressed after 12 h of induction, with an increasing yield over the course of the induction. Its maximum concentration was 7.83 mg/L after concentration and purification. In addition, in vitro experiments demonstrated that Cecropin P1 not only exerted a strong inhibitory effect on Escherichia coli, Salmonella sp., Shigella sp., and Pasteurella sp., but also displayed an antiviral activity against PRRSV NADC30-Like strain. Conclusions: Collectively, the strategy of expressing Cecropin P1 in Saccharomyces cerevisiae is harmless, efficient, and safe for cells. In addition, the expressed Cecropin P1 has antiviral and antibacterial properties concurrently.How to cite: Jiang R, Zhang P, Wu X, et al., Expression of antimicrobial peptide Cecropin P1 in Saccharomyces cerevisiae and its antibacterial and antiviral activity in vitro. Electron J Biotechnol 2021;50. https://doi.org/10.1016/j.ejbt.2020.12.006

This work was aimed to study the opportunity of transgenic oilseed rape (Brassica napus L.), expressing the gene of antimicrobial peptide cecropin P1 (cecP1) to be inhabited with associative microorganisms Methylobacterium mesophilicum and Pseudomonas aureofaciens. Previously, resistance was demonstrated to the microbial pathogens:Erwinia carotovora B15, Pseudomonas syringae, Fusarium oxysporum, and Botrytis cinerea. Analysis of the fatty acid composition of transgenic plant seeds showed an increase in the proportion of unsaturated fatty acids. Plants studied showed increased photosynthetic activity in oxidative stress induced by paraquat instead of in oxidative stress and UV radiation compared to control ones. A study of the interaction of transgenic plants with the associative microorganisms showed that bacteria were located in all initially colonized plants, as well as in plants obtained after several passages of microproliferation. Accordingly, transgenic plants, containing the cecP1 gene were actively colonized by associative bacteria, indicating their safety for associative bacteria, indicating their safety for beneficial microorganisms.

This work was aimed to study the opportunity of transgenic oilseed rape (Brassica napus L.), expressing the gene of antimicrobial peptide cecropin P1 (cecP1) to be inhabited with associative microorganisms Methylobacterium mesophilicum and Pseudomonas aureofaciens. Previously, resistance was demonstrated to the microbial pathogens:Erwinia carotovora B15, Pseudomonas syringae, Fusarium oxysporum, and Botrytis cinerea. Analysis of the fatty acid composition of transgenic plant seeds showed an increase in the proportion of unsaturated fatty acids. Plants studied showed increased photosynthetic activity in oxidative stress induced by paraquat instead of in oxidative stress and UV radiation compared to control ones. A study of the interaction of transgenic plants with the associative microorganisms showed that bacteria were located in all initially colonized plants, as well as in plants obtained after several passages of microproliferation. Accordingly, transgenic plants, containing the cecP1 gene were actively colonized by associative bacteria, indicating their safety for associative bacteria, indicating their safety for beneficial microorganisms. [ABSTRACT FROM AUTHOR]

Abstract Background In the past years, our laboratory successfully generated transgenic rainbow trout bearing cecropin P1 transgene. These fish exhibited resistant characteristic to infection by Aeromonas salmonicida, Infectious Hematopoietic Necrosis Virus (IHNV) and Ceratomyxa shasta (a parasitic pathogen). Previously, treating rainbow trout macrophage cells (RTS-11) with cecropin B, pleurocidin and CF17, respectively, resulted in elevated expression of two pro-inflammatory genes, e.g. cyclooxygenase-2 (cox-2) and interleukin-1β (il-1β). In addition, a profiling of global gene expression by 44 k salmonid microarray analysis was conducted, and the results showed that immune relevant processes have been perturbed in cecopin P1 transgenic rainbow trout. Therefore, we hypothesized that cecropin P1 may not only eliminate pathogens directly, but also modulate the host immune systems, leading to increased resistance against pathogen infections. To confirm this hypothesis, we performed de novo mRNA deep sequencing (RNA-Seq) to analyze the transcriptomic expression profiles in three immune competent tissues of cecropin P1 transgenic rainbow trout. Results De novo sequencing of mRNA of the rainbow trout spleen, liver and kidney tissues were conducted by second-generation Illumina system, followed by Trinity assembly. Tissue specific unigenes were obtained, and annotated according to the Gene Ontology (GO) and the Nucleotide Basic Local Alignment Search Tool (BLAST). Over 2000 differentially expressed genes (DEGs) were determined by normalized ratio of Reads Per Kilobase of transcript per million mapped reads (RPKM) among the transgenic and non-transgenic fish in a tissue specific manner, and there were 82 DEGs in common among the three tissues. In addition, the enrichment analysis according to Gene Ontology Biological Process (GO:BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) based pathway analysis associated with innate/adaptive immunity of fish were also performed to illustrate the altered immune-related functions in each tissue. Conclusions According to the RNA-Seq data, the correlations between alteration of gene expression profiles and the functional perturbations of the host immune processes were revealed. In comparison with the results of cDNA microarray analysis conducted by Lo et al., the overall results supported our hypothesis that the gene product of cecropin P1 transgene may not only directly eliminate pathogens, but also modulate the host immune system. Results of this study present valuable genetic information for Oncorhynchus mykiss, and will benefit future studies on the immunology of this fish species.

A sandwich immunoassay was developed for determination of E. coli O157:H7. This is based on an antimicrobial peptide-mediated nanocomposite pair and uses a personal glucose meter as signal readout. The antimicrobial peptides, magainins I, and cecropin P1 were employed as recognition molecules for the nanocomposite pair, respectively. With a one-step process, copper phosphate nanocomposites embedded by magainins I and Fe3O4 were used as "capturing" probes for bacterial magnetic isolation, and calcium phosphate nanocomplexes composed of cecropin P1 and invertase were used as signal tags. After magnetic separation, the invertase of the signal tags hydrolyzed sucrose to glucose, thereby converting E. coli O157:H7 levels to glucose levels. This latter can be quantified by a personal glucose meter. Under optimal conditions, the concentration of E. coli O157:H7 can be determined in a linear range of 10 to 107 CFU·mL−1 with a detection limit of 10 CFU·mL−1. The method was successfully applied to the determination of E. coli O157:H7 in milk samples. [ABSTRACT FROM AUTHOR]

Objective To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

Marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed with an agrobacterial binary vector free of selective genes of antibiotic and herbicide resistance. The marker-free transformants were screened via measurement of the antibacterial activity of cecropin P1 and enzyme immunoassay. The obtained plants exhibited an increased resistance to infection with the bacteria Erwinia carotovora, the fungi Fusarium graminearum, and oxidative stress during infection. Analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic Camelina lines as compared to unmodified plants. The results indicate that the cecropin P1 gene can be included in an integral antistress plant-protective system. [ABSTRACT FROM AUTHOR]

Recently, our laboratory had produced five families of transgenic rainbow trout harboring cecropin P1 transgene, and via repeated challenge studies these fish exhibited a significant elevation of resistance to infection by microbial pathogens. By cDNA microarray and mRNA deep sequencing (mRNA-seq) analyses on two of the five families of cecropin P1 transgenic fish, differentially expressed genes (DEGs) relevant to the innate and adaptive immune pathways in three different immune-related tissues, (i.e. spleen, kidney and liver) were profiled. These results supported our hypothesis that in addition to its direct microbicidal activity, the transgene product of cecropin P1 induces immunomodulatory activity in the transgenic host. Here, we have adapted the technique of quantitative reverse transcription real time PCR (RT-qPCR) array to analyze the expression of genes relevant to the innate and adaptive immune pathways in the rest three families. A RT-qPCR array was constructed with oligonucleotide primers of fifty-two innate/adaptive immune relevant DEGs shown to be the most perturbed by cecropin P1 transgene product in previous studies. Messenger RNA isolated from the spleen, kidney and liver of transgenic fish and non-transgenic fish control were studied on this array. Results of RT-qPCR array revealed that statistically significant perturbations of gene expression were detected in pathways of cytokine/chemokine signaling, Toll-like receptor signaling, complement cascade, antigen processing/presentation, lysosomal phagocytosis and leukocyte trans-endothelial migration in the transgenic spleen; extracellular matrix (ECM) organization and leukocyte trans-endothelial migration pathways in the transgenic kidney; lysosomal activity pathway in the transgenic liver. Furthermore, genes related to the pathways of the peroxisome proliferator-activated receptors (PPAR) signaling, lipid metabolism process and arachidonic acid metabolism were also impacted in the transgenic liver. Findings of the current study are in good agreement with those discoveries in previous two transgenic families by cDNA microarray and mRNA-seq analyses. • Cecropin P1 transgene product modulates the innate/adaptive immunity in the transgenic rainbow trout. • Transgenic spleen exhibited functional perturbations in a broad-range of immune relevant pathways. • Transgenic kidney exerted suppressions in leukocyte trans-endothelial migration and ECM organization. • Transgenic liver wielded functional disturbance in the immune pathway of lysosomal phagocytosis and alterations to energy efficiency related processes. • Custom-made RT-qPCR array provides a new, fast, and lower in cost screening method to select and breed high disease resistant strains of marine species. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Dye leakage from DMPC liposomes exposed to melittin and cecropin P1 (CP1) measured. • Lag time was in the order melittin I17K > melittin G1I > melittin > CP1. • Initial rate of dye leakage was in the reverse order of lag time. • Liposome size increased due to stretching caused by pore formation. • At high concentration, CP1 solubilized lipids suggesting carpet mechanism. Abstract Antimicrobial peptides are one of the most promising alternatives to antibiotics for targeting pathogens without developing resistance. In this study, pore formation in 1,2-Dimyristoyl-snglycero-3-phosphocholine (DMPC) / cholesterol liposome induced by native melittin, its two mutant variants (G1I and I17 K), and cecropin P1 was investigated by monitoring the dynamics of fluorescence dye leakage. A critical peptide concentration was required for dye leakage with the rate of leakage being dependent on peptide concentration above a critical value. A lag time was required for dye leakage for low peptide concentrations that are above the critical value, which decreased at higher peptide concentrations eventually approaching zero. Lag time was found to be in the order I17 K mutant with lower hydrophobicity and higher net charge > G1I with higher hydrophobicity > melittin > cecropin P1. Cecropin P1 exhibited the highest rate of dye leakage followed by melittin, G1I, and I17 K. Size distribution and transmission electron microscopy (TEM) of liposomes exposed to peptides of different concentrations indicated pore formation with accompanied stretching of liposomes at low peptide concentrations for both melittin and cecropin P1. At much higher concentrations, however, size distribution indicated three peaks for both peptides. In both cases, TEM images show that the middle and small peaks are shown to be due to stretched liposome and broken stretched liposome respectively. For melittin, the large peak is due to peptide aggregates as well as aggregates of liposome. For cecropin P1, however, the large peak indicates cecropin P1 aggregates with solubilized lipids thus suggesting carpet mechanism. [ABSTRACT FROM AUTHOR]

The method of producing of water extract from transgenic Kalanchoe pinnata L. plants expressing cecropin P1 gene has been developed and adapted for pharmaceutical production. Chlorophorm has been shown to be an efficient preservative for this extract. The antibiotic activity of the extract has kept well even after 1-h boiling. In addition, the prolonged antimicrobial activity of the extract has been confirmed by the "accelerated aging" method. Being added to culture medium, the leaf extract from transgenic K. pinnata plants has provided more intensive callus growth and improved rhizogenesis in ice plant (Mesembryanthemum crystallinum). The obtained results demonstrate an increased biological activity of the extract from kalanchoe plants producing antimicrobial cecropin P1 peptide and good prospects for its use in pharmacology. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) have the ability to penetrate the cell membrane, form pores which eventually lead to cell death. Immobilization of AMP on nanoparticles can play a major role in antimicrobial materials, biosensors for pathogen detection and in food safety. The minimum inhibitory concentration (MIC) of free Cecropin P1 (CP1, sequence SWLSTAKKLENSAKKRLSEGIAIAIQGGPR) and adsorbed on silica nanoparticle against E. coli O157:H7 EDL933 were 0.78 μg/ml. This was found to be consistent with preservation of α-helical secondary structure of CP1 upon adsorption as indicated by circular dichroism (CD). Cysteine-terminus modified Cecropin P1 (CP1C, sequence SWLSTAKKLENSAKKRLSEGIAIAIQGGPR C ) was chemically immobilized onto silica nanoparticles with maleimide-PEG-NHS ester cross-linkers of different PEG chain lengths. The antimicrobial activity of CP1C in solution and adsorbed on silica nanoparticles against E. coli O157:H7 EDL933 were found to be the same as those for CP1. However, tethered CP1C exhibited much higher MIC of 24.38, 37.55 and 109.82 μg/ml for (PEG) 20 , (PEG) 6 and (PEG) 2 linkers respectively. The antimicrobial activity of CP1C tethered to silica nanoparticles with (PEG) 20 linker was found to be lower for lower surface coverage with MIC values being 86.06, 36.89, 24.38 and 17.84 μg/ml for surface coverage of 12.3%, 24.4%, 52.8% and 83.8% respectively. All atom MD simulation of 1:3 DOPG/DOPC mixed membrane interacting with free and PEGlyated CP1C indicated that presence of PEG linker prevented CP1C from interacting with the bilayer which may explain the loss of antimicrobial activity of tethered CP1C. [ABSTRACT FROM AUTHOR]

Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptideexpressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/ CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and grampositive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis. [ABSTRACT FROM AUTHOR]

Accumulated evidence indicates that antimicrobial peptides modulate immune activities in fish. In this study, we profiled the differential expression patterns of representative immune relevant genes in an epithelial-like cell line of rainbow trout gill, RTgill-W1, in response to exposure of cecropin P1 antimicrobial peptide. RTgill-W1 cells were treated with synthetic cecropin P1 over time (0, 2, 4 and 24 h) with or without the present of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). The relative abundances of each mRNA were measured by real-time quantitative PCR. The dose-response study revealed significant perturbations of mRNA levels of genes related to pro-inflammation, acute phase, surface proteins and transcription factors at 30 μM of cecropin P1. In addition, cecropin P1 altered the differential expression patterns that were induced by LPS or PolyI:C, at different time points in RTgill-W1. Overall, our results indicate that cecropin P1 exhibits pro-inflammation activity, modulate cell-cell interaction and cytokine signal transduction in rainbow trout gill cell, and may suggest a potential application of this peptide as an immune adjuvant for disease control in aquaculture. • Utilizing RTgill-W1 cell line to build an in vitro model to represent the function of gill organ, studying differential expression profiles of immune relevant genes. • Cecropin P1 exerts pro-inflammatory effects and enhances inductions from LPS and PolyI:C treatments on RTgill-W1 cells. • Results provide better understandings of gill mucosal immunity, and may lead to development of novel applications that improve disease control in aquaculture production of rainbow trout. [ABSTRACT FROM AUTHOR]

Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad-spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram-negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram-negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α-helical structure in a solution containing LPS. For NMR experiments, we expressed 15N-labeled and 13C-labeled CP1 in bacterial cells and successfully assigned almost all backbone and side-chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr-NOE) experiments in LPS. We performed 15N-edited and 13C-edited Tr-NOE spectroscopy for CP1 bound to LPS. Tr-NOE peaks were observed at the only C-terminal region of CP1 in LPS. The results of structure calculation indicated that the C-terminal region (Lys15-Gly29) formed the well-defined α-helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Kalanchoe pinnate ( Kalanchöe pinnata L. ) plants with synthetic gene of antimicrobial peptide cecropin P1 (CP1) under the control of promoter 35S RNA of cauliflower mosaic virus (CaMV 35S) were produced. For transformation, a modified binary vector not containing selective genes of tolerance against antibiotics and herbicides was used. Screening of the marker-free transformed plants was conducted on the medium without selective antibiotics by revealing antibacterial activity of plant extracts and cecropin P1. The marker-free plants produced displayed increased resistance against bacterial and fungus phytopathogens, while their extracts were characterized by antimicrobial activity for human and animal pathogens. These plants meet the requirements of biosafety and may be used as producers of cecropin P1 in pharmaceutics. [ABSTRACT FROM AUTHOR]

A gene of antimicrobial peptide cecropin P1 ( CP1) was inserted into the vector plasmid pPCV91 under the control of promoter 35S RNA of cauliflower mosaic virus (CaMV 35S) containing four enhancer sequences CaMV 35S and nontranslated leader sequence Ω RNA of tobacco mosaic virus. The recombinant vector obtained was used for agrobacterial transformation of tobacco plants ( Nicotiana tabacum L., variety Samsun) with the polymerase chain reaction (PCR)-based method. The presence of gene CP1 in the genome of plants was proven by western-blot analysis and testing the antibiotic activity of plant extracts. In different plant lines, the level of cecropin P1 synthesis amounted 0.02-0.2% of total soluble plant leaf protein. The transgenic plants, unlike the control ones, displayed enhanced tolerance to phytopathogenic microorganisms and oxidative stress. It was established that the ability of the transgenic plants to express cecropin P1 is transmitted to progeny. [ABSTRACT FROM AUTHOR]

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pig industry, causing high economic losses worldwide. Current vaccines have specific limitations in terms of their safety and efficacy, so the development of novel antiviral drugs is urgently required. The aim of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of the antimicrobial peptide cecropin P1 (CP1) against PRRSV infection in vitro. Results: CP1 not only displayed extracellular virucidal activity against PRRSV, but also exerted a potent inhibitory effect when added either before, simultaneously with, or after viral inoculation. The inhibitory effect of CP1 occurred during viral attachment, but not at viral entry into Marc-145 cells. CP1 also inhibited viral particle release and attenuated virus-induced apoptosis during the late phase of infection. CP1 exerted similar inhibitory effects against PRRSV infection in porcine alveolar macrophages, the cells targeted by the virus in vivo during its infection of pigs. The expression of interleukin 6 was elevated by CP1 in porcine alveolar macrophages, which might contribute to its inhibition of PRRSV infection. Conclusions: Collectively, our findings provide a new direction for the development of potential therapeutic drugs against PRRSV infection. [ABSTRACT FROM AUTHOR]

Previous studies conducted in our laboratory showed that transgenic medaka expressing cecropin B transgenes exhibited resistant characteristic to fish bacterial pathogens, Pseudomonas fluorescens and Vibrio anguillarum. To confirm whether antimicrobial peptide gene will also exhibit anti-bacterial and anti-viral characteristics in aquaculture important fish species, we produced transgenic rainbow trout expressing cecropin P1 or a synthetic cecropin B analog, CF-17, transgene by sperm-mediated gene transfer method. About 30 % of fish recovered from electroporation were shown to carry the transgene as determined by polymerase chain reaction (PCR) amplification assay. Positive P transgenic fish were crossed to non-transgenic fish to establish F transgenic founder families, and subsequently generating F, and F progeny. Expression of cecropin P1 and CF-17 transgenes was detected in transgenic fish by reverse transcription (RT)-PCR analysis. The distribution of body sizes among F transgenic fish were not significantly different from those of non-transgenic fish. Results of challenge studies revealed that many families of F and F transgenic fish exhibited resistance to infection by Aeromonas salmonicida and infectious hematopoietic necrosis virus (IHNV). All-male homozygous cecropin P1 transgenic families were produced by androgenesis from sperm of F heterozygous transgenic fish in one generation. The resistant characteristic to A. salmonicida was confirmed in progeny derived from the outcross of all-male fish to non-transgenic females. Results of our current studies confirmed the possibility of producing disease-resistant homozygous rainbow trout strains by transgenesis of cecropin P1 or CF-17 gene and followed by androgenesis. [ABSTRACT FROM AUTHOR]

Abstract: The binding capacity of peptides with broad antimicrobial activity, or antimicrobial peptides (AMPs), to microbes has recently been applied to the specific detection of bacteria and viruses. We established a novel lateral flow assay (LFA) that combines AMPs labeled with colloidal gold and a target-specific antibody immobilized on a nitrocellulose membrane. α-Helical AMPs, especially cecropin P1 (CP1), magainin 2 (MG2), and ceratotoxin A (CtxA), were shown to have optimal properties as probes in LFA. We also established a multiplex LFA for the simultaneous detection and identification of three serogroups of Shiga toxin-producing Escherichia coli (STEC) using the CP1 probe with polyclonal antibodies anti-O157, anti-O26, and anti-O111. Each serogroup of E. coli could easily and rapidly be detected by multiplex LFA using CP1 and each was clearly visualized in a different position on the LFA strip. The multiplex LFA could detect all tested E. coli strains from serogroups O157 (22/22), O26 (17/17), and O111 (7/7), and the detection limit was 104 CFU/mL. No other serogroups of E. coli, including STEC O45, O91, O103, O121, and O145, or non-E. coli strains, reacted. The multiplex LFA could detect E. coli O157, O26, and O111 in food samples at very low levels (6.3, 2.9, and 5.6CFU per 25g of ground beef, respectively) after 18-h enrichment, and these results were in accordance with the results of the culture method, immunochromatography (IC) strip, and PCR. Given the broad binding capacity, AMP probes in combination with specific antibodies in the novel multiplex LFA may have the potential to detect various microbes simultaneously with identification on a single strip. [Copyright &y& Elsevier]

Transgenic rapeseed ( Brassica napus L.) plants carrying an artificial gene for the antimicrobial peptide cecropin P1 ( cecP1) were obtained and characterized. The agrobacterial transformation was done by vacuum infiltration of seeds with agrobacterium GV3101(pMP90RK) containing a binary vector pGA482:: cecP1. The cec1 gene expression was analyzed by Western blotting and confirmed by antimicrobial activity measurements of plant extracts. The obtained plants showed the resistance to the bacterial and fungal pathogens Erwinia carotovora and Fusarium sporotrichioides. The photosynthetic activities of control and transgenic plants under biotic stress conditions of E. carotovora infection were comparatively studied. The higher tolerance of the cecP1 plants to the oxidative stress caused by paraquat was shown. The results obtained point to the possibility of incorporation of the cecropin P1 gene into the integral stress protection system of plants. [ABSTRACT FROM AUTHOR]

Cecropins are a group of antimicrobial peptides which have bactericidal activity against a broad range of bacteria. To date, the cecropins used in a variety of studies were either purified from their natural source or obtained by chemical synthesis. The present study was conducted to test whether bactericidally active cecropins could be expressed in a fish cell line. For this purpose, Chinook salmon embryo cells (CHSE-214) were transfected with cecropin transgene constructs: Hyalophora cecropia preprocecropin B, procecropin B, cecropin B, and porcine P1 cecropin. From the transfected cells, single cell clones were selected and screened for the presence of cecropin gene constructs by PCR amplification. The expression of the cecropin transgene in the PCR positive clones was determined by RT-PCR reaction. Southern blot hybridization results showed that the cecropin gene constructs were integrated into the genome in a multiple integration pattern. Bactericidal activity of the cecropins, synthesized from transgene constructs, was detected using inhibition zone assay for fish pathogenic bacteria: Aeromonas hydrophila, Pseudomonas fluorescens, and Vibrio anguillarum. Cecropin antimicrobial peptides produced in CHSE-214 cells possess bactericidal activity against these three fish pathogenic bacteria. [Copyright &y& Elsevier]

Organisms co-habiting with bacteria have developed efficient bactericidal agents to control their microbe-rich environment. The Ascaris nematode lives in its final development stages in the gut of its host and is believed to feed on bacteria. Ascaris suum survive in pig intestine while A. lumbricoides is the principal species in humans. Here we show that A. suum and A. lumbricoides both produce linear (cecropin P1) and cysteine-rich (ASABF) peptides with activity against either Gram-negative or Gram-positive bacteria, respectively. Thus nematodes rely in part on a peptide-based antibacterial system for digestion of bacteria, which may also confer protection against infection. Cecropin P1 was previously isolated from pig intestine but we can now conclude that was due to contaminating nematodes. [ABSTRACT FROM AUTHOR]