Identification and characterization of a novel antibacterial peptide,avian β-defensin 2 from ducks

In this study, a novel avian β-defensin (AvBD) was isolated from duck pancreas. The complete nucleotide sequence of the gene contained an 195 bp open reading frame encoding 64 amino acids. Homology, characterization and comparison of the gene with AvBD from other avian species confirmed that it was duck AvBD2. The mRNA expression of the gene was analyzed in 17 tissues from 21-day-old ducks. AvBD2 was highly expressed in the trachea, crop, heart, bone marrow, and pancreas; moderately expressed in the muscular stomach, small intestine, kidney, spleen, thymus, and bursa of Fabricius; and weakly expressed in skin. We produced and purified recombinant AvBD2 by expressing the gene in Escherichia coli. As expected, the recombinant peptide exhibited strong bactericidal properties against Bacillus cereus, Staphylococcus aureus, and Pasteurella multocida, and weak bactericidal properties against E. coli and Salmonella choleraesuis. In addition, the recombinant protein retained antimicrobial activity against S. aureus under different temperatures (range, −20°C to 100°C) and pH values (range, 3 to 12)     

鸽β-防御素1基因的克隆及其生物学作用鉴定

【目的】从鸽子组织中克隆鸽子β-防御素1(AvBD1)基因,在大肠杆菌中表达重组鸽子AvBD1蛋白,测定其生物学特性。【方法】应用RT-PCR法从鸽子骨髓组织中扩增鸽子AvBD1基因,采用Real-time PCR法检测该基因在鸽子组织器官中的表达分布。将该基因亚克隆到大肠杆菌原核表达载体pProEX-HTa的EcoR I和Xho I双酶切位点上,构建重组表达质粒pProEX-pigeon AvBD1,将重组质粒进行诱导表达;对该重组蛋白进行纯化,通过菌落计数法测定其体外抗菌活性与理化特性。【结果】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其cDNA大小为198 bp,编码65个氨基酸,经序列相似性分析,鸽子AvBD1与鸭AvBD1氨基酸序列相似性最高(81.5%)。鸽子AvBD1主要分布于免疫系统和消化系统组织中。Tricine-SDS-PAGE电泳结果表明,重组鸽子AvBD1蛋白分子量约8.8 kD,与预期大小一致。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性。此外,该重组蛋白的溶血活性极低。【结论】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其主要分布在机体的免疫系统和消化系统中。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性,且该重组蛋白的溶血活性极低。     

Synergistic activity of Bacillus thuringiensis δ‐endotoxin and TMOF against Culex pipiens and Spodoptera littoralis larvae

Trypsin Modulating Oostatic Factor (TMOF) is a decapeptide hormone that inhibits the biosynthesis of digestive enzymes in the mosquito midgut. The hormone inhibits food digestion and ultimately leads to starvation and death. It has been used as a biological insecticide to control mosquitoes. In an attempt to increase the insecticidal activity of TMOF, a combination of CryIC (δ‐endotoxin from Bacillus thuringiensis) and TMOF was determined. Eight recombinant proteins fused with GST (glutathione‐S‐transferase) were expressed in Escherichia coli cells. Their insecticidal activities were determined against Culex pipiens and Spodoptera littoralis larvae. Purified GST‐TMOF and its analogue GST‐YDPAS exhibited a moderate toxicity on C. pipiens larvae with LC₅₀ of 145.9 and 339.9 μg/mL, respectively. Unexpectedly, no mortality was observed in first instar larvae of S. littoralis. Puirified GST‐TMOF and GST‐YDPAS together with Bt toxin showed a synergistic toxic effect on both Culex and Spodoptera larvae. In the presence of 100 μg/mL GST‐TMOF and GST‐YDPAS, the median lethal concentration of entomocidus on culex larvae decreased from 52.1 to 16.7 and 31.9 μg/mL, respectively. Likewise, GST‐TMOF and GST‐YDPAS incorporated with 0.07 μg/cm² of enotmocidus showed insecticidal activity against S. littoralis with LC₅₀ of 16.4 and 21.9 μg/cm². The E. coli lysates containing GST‐CryIC and its 3′‐truncated version showed low toxicity against the lepidopteran insect (10.8 and 16.6 μg/cm²) compared to 0.15 μg/cm² of the native crystalline form of CryIC. Similarly, the mosquitocidal activity of the recombinant Bt toxins was low.     

Prokaryotic expression and antimicrobial mechanism of XPF‐St7‐derived α‐helical peptides

XPF‐St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α‐helical segment of XPF‐St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram‐negative and Gram‐positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6‐fold and 6.7‐fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

鹅β-防御素3基因的分离、鉴定及其表达产物的生物学特性

为了克隆鹅β-防御素(AvBD)3基因,并在原核表达重组鹅AvBD3蛋白,进一步研究鹅AvBD3蛋白的生物学特性,利用RT-PCR方法从鹅脾脏和法氏囊组织中扩增到鹅AvBD3基因片段,其cDNA片段大小为182 bp,编码60个氨基酸残基.经同源性分析发现鹅AvBD3氨基酸序列与鸡AvBD3氨基酸序列同源性最高,为100％.将该基因亚克隆到原核表达载体pGEX-6p-1的BamH Ⅰ和SalⅠ双酶切位点上,构建重组表达质粒pGEX-goose AvBD3.将重组质粒转化大肠杆菌BL21,于37℃用IPTG诱导表达,SDS-PAGE电泳表明,重组鹅AvBD3蛋白在原核高效表达(分子量约31 kDa).该重组蛋白经纯化后测定其体外抗菌活性与理化特性,结果显示,重组鹅AvBD3蛋白具有广谱的抗菌活性,对12种细菌,包括革兰氏阳性菌和革兰氏阴性菌均具有抑菌作用.高盐离子浓度显著降低重组鹅AvBD3蛋白的抗菌活性.此外,该重组蛋白的溶血活性极低,并对酸碱度具有较高的稳定性.     

Haplotype structure and copy number polymorphism of the beta‐defensin 7 genes in diverse chicken breeds

Beta‐defensins is a family of avian peptides related to the innate immune system. Copy number variation was recently reported for the avian beta‐defensin 7 gene (AvBD7) between the highly inbred Leghorn and Fayoumi lines. Here, we examined copy number variants in 35 different chicken breeds and found that 31 of them have at least the same representation of the duplicated AvBD7 allele. We also found haplotypes upstream of the AvBD6 regions that are strongly linked to the AvBD7 duplication. We observed a strong linkage disequilibrium spanning of the upstream region of the AvBD6 gene, with two SNPs being flanking markers to detect duplication of the AvBD7.     

High‐yield recombinant expression of the chicken antimicrobial peptide fowlicidin‐2 in Escherichia coli

The antimicrobial peptide fowlicidin‐2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin‐2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin‐2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin‐2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET‐32a(+), which features fusion protein thioredoxin at the N‐terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria‐Bertani (LB) medium. After isopropyl‐β‐D‐thiogalactopyranoside (IPTG) induction, the fowlicidin‐2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse‐phase high‐performance liquid chromatography (RP‐HPLC), ～6.0 mg of fowlicidin‐2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram‐positive and Gram‐negative bacteria, and even drug‐resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large‐scale production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:369–374, 2015     

Antifungal activity of recombinant mouse beta‐defensin 3

Aims: To identify the presence of mouse β‐defensin 3 (Mbd3) (the human homologue of β‐defensin 2) in different tissues and to define the antimicrobial properties of recombinant MBD3 (rMBD3) against a panel of human pathogens. Methods and Results: Mbd3 gene expression in different mouse tissues before or after lipopolysaccharide (LPS) injection was compared by semi‐quantitative RT‐PCR. This analysis demonstrated that epithelial and mucosal tissues expressed Mbd3 independent of LPS stimulation. Evaluation of the antimicrobial properties of recombinant rMBD3 was determined by assessing the median inhibition concentration (IC₅₀), minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)/minimal fungicidal concentration (MFC) against various human pathogens. Conclusion: Mbd3 gene expression by epithelial and mucosal tissues suggested that MBD3 likely plays an early defensive role against microbial infections. This activity was most significant against filamentous fungi. Significance and Impact of the Study: The data presented in this report suggested that formulations containing rMBD3 and related molecules could serve to treat fungal and bacterial infections.     

Defensin‐like peptide3 from black solder fly: Identification,characterization, and key amino acids for anti‐Gram‐negative bacteria

An antibacterial peptide was isolated from a black soldier fly, Hermetia illucens. The molecular mass of this peptide was established as 4247.37 by matrix‐assisted laser desorption/ionization‐time of flight mass (MALD‐TOF MS) spectrometry. The amino acid sequence of the mature peptide was determined by N‐terminal sequencing using Edman degradation, combined with cDNA sequencing of the previously reported defensin‐like peptide (DLP) 3. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP3 had potent activity against Gram‐positive and negative bacteria, but DLP4 had only anti‐Gram‐positive activity as previously reported. Recombinant DLP3 and DLP4 were overexpressed in Escherichia coli, and antibacterial activities were identical to DLPs purified from H. illucens hemolyph. In silico analysis revealed that only six amino acid sequences were different between DLP3 and DLP4, but antibacterial activity against Gram‐negative bacteria differed. Therefore these amino acid variants may be key amino acids (Gly‐10, Val‐18, Met‐23, Arg‐25, Asp‐32, Arg‐40) related to killing Gram‐negative bacteria.     

Antimicrobial activity of peptides derived from human ß‐amyloid precursor protein

Antimicrobial peptides are important effector molecules of the innate immune system. Here, we describe that peptides derived from the heparin‐binding disulfide‐constrained loop region of human ß‐amyloid precursor protein are antimicrobial. The peptides investigated were linear and cyclic forms of NWCKRGRKQCKTHPH (NWC15) as well as the cyclic form comprising the C‐terminal hydrophobic amino acid extension FVIPY (NWCKRGRKQCKTHPHFVIPY; NWC20c). Compared with the benchmark antimicrobial peptide LL‐37, these peptides efficiently killed the Gram‐negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram‐positive Staphylococcus aureus and Bacillus subtilis, and the fungi Candida albicans and Candida parapsilosis. Correspondingly, fluorescence and electron microscopy demonstrated that the peptides caused defects in bacterial membranes. Analogously, the peptides permeabilised negatively charged liposomes. Despite their bactericidal effect, the peptides displayed very limited hemolytic activities within the concentration range investigated and exerted very small membrane permeabilising effects on human epithelial cells. The efficiency of the peptides with respect to bacterial killing and liposome membrane leakage was in the order NWC20c > NWC15c > NWC15l, which also correlated to the adsorption density for these peptides at the model lipid membrane. Thus, whereas the cationic sequence is a minimum determinant for antimicrobial action, a constrained loop‐structure as well as a hydrophobic extension further contributes to membrane permeabilising activity of this region of amyloid precursor protein. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Structural and functional characterization of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy‐terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os–C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94–15 µg/ml) to both Gram‐positive and Gram‐negative bacteria, whereas the parent peptide only exhibited Gram‐positive antibacterial activity. The Os peptide was found to be two‐fold more active than Os–C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os–C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane‐mimicking environment, the cysteine‐containing peptide has a higher α‐helical content. Both peptides were found to be non‐toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial function of short amidated peptide fragments from the tick‐derived OsDef2 defensin

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram‐positive and Gram‐negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3–12) and Os(11–22) exhibit activity when screened against Gram‐positive and Gram‐negative bacteria. Carboxyamidation of both peptides increased membrane‐mediated activity, although carboxyamidation of Os(11–22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3–12)NH₂ and Os(11–22)NH₂, have minimum bactericidal concentrations of 3.3 μM against Escherichia coli. Killing was reached within 10 minutes for Os(3–12)NH₂ and only during the second hour for Os(11–22)NH₂. In an E. coli membrane liposome system, both Os and Os(3–12)NH₂ were identified as membrane disrupting while Os(11–22)NH₂ was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3–12)NH₂ did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3–12 fragment relies on a membrane disruptive mechanism while the 11–22 fragment involves additional target mechanisms. The salt‐resistant potency of Os(3–12)NH₂ identifies it as a promising candidate for further development.     

Purification of glutathione S‐transferase enzyme from quail liver tissue and inhibition effects of (3aR,4S,7R,7aS)‐2‐(4‐((E)‐3‐(aryl)acryloyl)phenyl)‐3a,4,7,7a‐tetrahydro‐1H‐4,7‐methanoisoindole‐1,3(2H)‐dione derivatives on the enzyme activity

The use of quail meat and eggs has made this animal important in recent years, with its low cost and high yields. Glutathione S‐transferases (GST, E.C.2.5.1.18) are an important enzyme family, which play a critical role in detoxification system. In our study, GST was purified from quail liver tissue with 47.88‐fold purification and 12.33% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by SDS‐PAGE method and showed a single band. In addition, inhibition effects of (3aR,4S,7R,7aS)‐2‐(4‐((E)‐3‐(aryl)acryloyl)phenyl)‐3a,4,7,7a‐tetrahydro‐1H‐4,7methanoisoindole‐1,3(2H)‐dion derivatives ( 1a–g ) were investigated on the enzyme activity. The inhibition parameters (IC₅₀ and K_i values) were calculated for these compounds. IC₅₀ values of these derivatives ( 1a–e ) were found as 23.00, 15.75, 115.50, 10.00, and 28.75 μM, respectively. K_i values of these derivatives ( 1a–e ) were calculated in the range of 3.04 ± 0.50 to 131.50 ± 32.50 μM. However, for f and g compounds, the inhibition effects on the enzyme were not found.     

Comparative <Emphasis Type="Italic">in vivo</Emphasis> infection models yield insights on early host immune response to Campylobacter in chickens

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian β-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bldesin,the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities

Fungus defensin is a kind of important natural peptide resource, such as plectasin from the soil fungus Pseudoplectania nigrella with potential application in the antimicrobial peptide lead drug discovery. Here, a fungus defensin named Bldesin with Kv1.3 channel and serine protease inhibitory activities was first explored. By GST‐Bldesin fusion expression and enterokinase cleaving strategy, recombinant Bldesin was obtained successfully. Antimicrobial assays showed that Bldesin had potent activity against Gram‐positive Staphylococcus aureus, but had no effect on Gram‐negative Escherichia coli. Electrophysiological experiments showed that Bldesin had Kv1.3 channel inhibitory activity. Serine protease inhibitory associated experiments showed that Bldesin had unique chymotrypsin protease inhibitory, elastase protease inhibitory, and serine protease‐associated coagulation inhibitory activities. To the best of our knowledge, Bldesin is the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities and highlighted novel pharmacological effects of fungus‐derived defensin peptides.