Broad activity against porcine bacterial pathogens displayed by two insect antimicrobial peptides moricin and cecropin B

In response to infection, insects produce a variety of antimicrobial peptides (AMPs) to kill the invading pathogens. To study their physicochemical properties and bioactivities for clinical and commercial use in the porcine industry, we chemically synthesized the mature peptides Bombyx mori moricin and Hyalophora cecropia cecropin B. In this paper, we described the antimicrobial activity of the two AMPs. Moricin exhibited antimicrobial activity on eight strains tested with minimal inhibitory concentration values (MICs) ranging between 8 and 128 μg/ml, while cecropin B mainly showed antimicrobial activity against the Gramnegative strains with MICs ranging from 0.5 to 16 μg/ml. Compared to the potent antimicrobial activity these two AMPs displayed against most of the bacterial pathogens tested, they exhibited limited hemolytic activity against porcine red blood cells. The activities of moricin and cecropin B against Haemophilus parasuis SH 0165 were studied in further detail. Transmission electron microscopy (TEM) of moricin and cecropin B treated H. parasuis SH 0165 indicated extensive damage to the membranes of the bacteria. Insights into the probable mechanism utilized by moricin and cecropin B to eliminate pathogens are also presented. The observations from this study are important for the future application of AMPs in the porcine industry.     

Insect antimicrobial peptides and their applications

Insects are one of the major sources of antimicrobial peptides/proteins (AMPs). Since observation of antimicrobial activity in the hemolymph of pupae from the giant silk moths Samia Cynthia and Hyalophora cecropia in 1974 and purification of first insect AMP (cecropin) from H. cecropia pupae in 1980, over 150 insect AMPs have been purified or identified. Most insect AMPs are small and cationic, and they show activities against bacteria and/or fungi, as well as some parasites and viruses. Insect AMPs can be classified into four families based on their structures or unique sequences: the α-helical peptides (cecropin and moricin), cysteine-rich peptides (insect defensin and drosomycin), proline-rich peptides (apidaecin, drosocin, and lebocin), and glycine-rich peptides/proteins (attacin and gloverin). Among insect AMPs, defensins, cecropins, proline-rich peptides, and attacins are common, while gloverins and moricins have been identified only in Lepidoptera. Most active AMPs are small peptides of 20–50 residues, which are generated from larger inactive precursor proteins or pro-proteins, but gloverins (~14 kDa) and attacins (~20 kDa) are large antimicrobial proteins. In this mini-review, we will discuss current knowledge and recent progress in several classes of insect AMPs, including insect defensins, cecropins, attacins, lebocins and other proline-rich peptides, gloverins, and moricins, with a focus on structural-functional relationships and their potential applications.     

家蚕丝氨酸蛋白酶抑制剂Bmserpin2对酚氧化酶原激活和抗菌肽基因表达的抑制作用

【目的】丝氨酸蛋白酶抑制剂家族蛋白是昆虫中调控自身免疫反应的重要蛋白酶抑制剂,本研究旨在研究家蚕Bombyx mori丝氨酸蛋白酶抑制剂2(Bmserpin2)在家蚕2个重要的自身免疫通路即酚氧化酶原(prophenol oxidase, PPO)激活通路和革兰氏阳性菌诱导抗菌肽的TOLL通路中的调控作用。【方法】PCR扩增家蚕Bmserpin2基因片段后原核表达并通过镍柱纯化。利用纯化后的重组Bmserpin2蛋白分别与胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶和蛋白酶K反应,检测Bmserpin2对上述蛋白酶活性的影响。通过RT-qPCR检测Bmserpin2在家蚕5龄第3天幼虫头、中肠、脂肪体、血淋巴、丝腺和表皮组织中表达的模式。往家蚕5龄第3天幼虫注射Bmserpin2重组蛋白,检测Bmserpin2对其血淋巴中PPO活性的影响。通过滕黄微球菌Micrococcus luteus诱导家蚕5龄第3天幼虫产生抗菌肽并注射Bmserpin2重组蛋白后,RT-qPCR检测其血淋巴中抗菌肽基因gloverin2和moricin表达量。【结果】成功构建重组质粒并表达纯化目的蛋白Bmserpin2。通过与不同蛋白酶反应得出Bmserpin2可极显著抑制消化酶胰蛋白酶和弹性蛋白酶活性,对胰凝乳蛋白酶和蛋白酶K活性影响不显著,提示Bmserpin2对不同蛋白酶具有生物学活性和催化特异性。基因表达模式显示Bmserpin2在家蚕5龄幼虫血淋巴和脂肪体中表达量最高。家蚕5龄幼虫注射重组Bmserpin2蛋白后发现目的蛋白能有效抑制血淋巴中PPO活性。利用滕黄微球菌诱导家蚕5龄幼虫产生抗菌肽后,滕黄微球菌和Bmserpin2混合注射组中血淋巴中抗菌肽基因gloverin2和moricin的转录表达与只注射滕黄微球菌的比较被显著下调。【结论】Bmserpin2可能参与家蚕酚氧化酶原激活和TOLL途径的胞外级联反应的免疫通路。     

Eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection

Phospholipase A(2) (PLA(2)) catalyzes hydrolysis of phospholipids at sn-2 position and usually releases arachidonic acid, which is oxygenated into various eicosanoids that mediate innate immune responses in insects. PLA(2) activities were measured in both immune-associated tissues of hemocyte and fat body in the beet armyworm, Spodoptera exigua. Upon challenge of an entomopathogenic fungus, Beauveria bassiana, the PLA(2)s were significantly activated in both hemocyte and fat body. The fungal infection also induced gene expression of antimicrobial peptides (AMPs), such as two attacins, cecropin, gallerimycin, gloverin, hemolin, and transferrin of S. exigua. RNA interference of Toll or Imd signal pathway using double-stranded RNAs (dsRNAs) specific to SeToll or SeRelish suppressed specific AMP gene expressions, in which dsRNA specific to SeToll suppressed two attacins, cecropin, gallerimycin, gloverin, hemolin, and transferrin I, while dsRNA specific to SeRelish suppressed only cecropin. Interestingly, dsRNA specific to SeToll also significantly inhibited the activation of PLA(2) in response to the fungal infection, but dsRNA specific to SeRelish did not. Eicosanoid-dependent hemocyte nodulation was inhibited by dsRNA specific to SeToll but was not by dsRNA specific to SeRelish. These results suggest that eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection in S. exigua.     

Purification and characterization of eight peptides from Galleria mellonella immune hemolymph

Defense peptides play a crucial role in insect innate immunity against invading pathogens. From the hemolymph of immune-challenged greater wax moth, Galleria mellonella (Gm) larvae, eight peptides were isolated and characterized. Purified Gm peptides differ considerably in amino acid sequences, isoelectric point values and antimicrobial activity spectrum. Five of them, Gm proline-rich peptide 2, Gm defensin-like peptide, Gm anionic peptides 1 and 2 and Gm apolipophoricin, were not described earlier in G. mellonella. Three others, Gm proline-rich peptide 1, Gm cecropin D-like peptide and Galleria defensin, were identical with known G. mellonella peptides. Gm proline-rich peptides 1 and 2 and Gm anionic peptide 2, had unique amino acid sequences and no homologs have been found for these peptides. Antimicrobial activity of purified peptides was tested against gram-negative and gram-positive bacteria, yeast and filamentous fungi. The most effective was Gm defensin-like peptide which inhibited fungal and sensitive bacteria growth in a concentration of 2.9 and 1.9 microM, respectively. This is the first report describing at least a part of defense peptide repertoire of G. mellonella immune hemolymph.     

家蚕拟抗微生物肽Gloverins基因（Bmglv）的原核表达及抗菌活性鉴定

家蚕Bombyx mori基因组全测序于2004年完成后,由家蚕基因组注释发现了35条拟抗微生物肽基因序列。本研究选取其中的5条Gloverins同系物（isoforms）基因(BmglvB,BmglvA2 ,BmglvA3,BmglvA5和BmglvA6 ),克隆至pET-21d载体,转化Escherichia coli Rosetta^TM（DE3）宿主菌进行表达;克隆表达的5个家蚕Gloverins同系物基因大部分以可溶形式表达,经Ni-NTA亲和层析纯化和Sephadex G-10脱盐处理后,采用平板孔穴抑菌法测定其抗菌活性。结果表明：注释发现的5条家蚕拟抗微生物肽Gloverins同系物基因,具有与在其他昆虫中已鉴定报道的Gloverin相似的抗革兰氏阴性细菌的功能,实验确认了它们就是家蚕Gloverins抗微生物肽基因。     

Primary structure and in vitro antibacterial properties of the Drosophila melanogaster attacin C Pro-domain

In Drosophila melanogaster, seven distinct families of antimicrobial peptides with different structures and specificities are synthesized by the fat body and released into the hemolymph during the immune response. Using microscale high performance liquid chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Edman degradation, we have isolated and characterized from immune-challenged Drosophila two novel induced molecules, under the control of the Imd pathway, that correspond to post-translationally modified antimicrobial peptides or peptide fragments. The first molecule is a doubly glycosylated form of drosocin, an O-glycosylated peptide that kills Gram-negative organisms. The second molecule represents a truncated form of the pro-domain of the Drosophila attacin C carrying two post-translational modifications and has significant structural similarities to proline-rich antibacterial peptides including drosocin. We have synthesized this peptide and found that it is active against Gram-negative bacteria. Furthermore, this activity is potentiated when the peptide is used in combination with the Drosophila antimicrobial peptide cecropin A. The synergistic action observed between these two molecules suggests that the truncated post-translationally modified pro-domain of attacin C by itself may play an important role in the antimicrobial defense of Drosophila.     

Geographically Distinct Expression Profile of Host Defense Peptides in the Skin of the Chinese Odorous Frog,Odorrana margaretae

Odorrana margaretae （Anura： Ranidae） is widely distributed in the southern provinces of China. Previously, 72 antimicrobial peptides （AMPs） belonging to 21 families were identified from the skin of O. margaretae, which were captured in the Hunan province. In the present study, five O. margaretae frogs were captured from the Guizhou province and a total of 28 cDNAs encoding 17 host defense peptides （HDPs） belonging to 14 families were cloned from the skin cDNA library of O. margaretae. Among the 17 HDPs, only one （brevinin-1-Omar5） had been characterized. The distinct HDP expression profiles for O. margaretae in the previous and present study may be attributed to the environmental differences between the sampling locations and the genetic divergence among O. margaretae populations. Besides, 11 of the 17 HDPs identified in the present study were novel for ranids. In order to understand their roles in host defense reactions, three HDPs （odorranain-H-OM1, odorranain-M-OM and ranatuerin-2-OM）, which possess low sequence similarity with the known amphibian HDPs, were selected for further chemical synthesis and functional analysis. Odorranain-H-OM1 showed direct antimicrobial activity against bacteria and fungi. Odorranain-M-OM exhibited concentration-dependent anti-oxidant activity. Ranatuerin-2-OM showed lectin-like activity and could strongly hemagglu -tinate human intact erythrocytes with or without the presence of Ca2＋. The diverse activities of HDPs implied that they may play different roles in host defense reactions of O. margaretae.     

斜纹夜蛾抗菌肽基因Moricin和Cecropin时空表达模式及微生物敏感性的分析

抗菌肽是一类具有广谱抗菌活性的小分子多肽, 包括抗细菌、 病毒和真菌。本研究以斜纹夜蛾Spodoptera litura (Fabricius)为研究对象, 利用Real-time PCR和半定量RT-PCR检测了其体内两个重要抗菌肽基因Cecropin和Moricin的时空表达模式及其对微生物的敏感性。Real-time PCR检测结果表明, 抗菌肽基因Cecropin和Moricin在斜纹夜蛾各个龄期（1, 2, 3, 4, 5和6龄幼虫、 蛹、 成虫）都有表达, 随着龄期的增长, 表达量逐渐增加, 6龄幼虫中表达量达到最高。 半定量RT-PCR检测表明, 抗菌肽基因Cecropin和Moricin在7个不同组织（气管、 卵巢、 马氏管、 体壁、 中肠、 脂肪体和血细胞）中都有表达, Cecropin在脂肪体中表达量最高, 而Moricin在血细胞中表达量相对较高; RT-PCR检测表明, Cecropin和Moricin的表达在大肠杆菌Escherichia coli诱导后迅速上升, 其中诱导后8 h达到高峰期, 一直持续到48 h。不同病原微生物（大肠杆菌E. coli、 金黄色葡萄球菌Staphyloccocus aureus和白僵菌Beauveria bassiana）分别诱导后, Real-time PCR 检测表明, 斜纹夜蛾抗菌肽基因Cecropin和Moricin对大肠杆菌的刺激最为敏感。本研究结果为进一步研究斜纹夜蛾抗菌肽基因Cecropin和Moricin的功能奠定基础。     

Design and analysis of structure-activity relationship of novel antimicrobial peptides derived from the conserved sequence of cecropin.

We have de novo designed four antimicrobial peptides AMP-A/B/C/D, the 51-residues peptides, which are based on the conserved sequence of cecropin. In the present study, the four peptides were chemically synthesized and their activities assayed. Their secondary structure, amphipathic property, electric field distribution and transmembrane domain were subsequently predicted by bioinformatics tools. Finally, the structure-activity relationship was analyzed from the results of activity experiments and prediction. The results of activity experiments indicated that AMP-B/C/D clearly possessed excellent broad-spectrum activity against bacteria, whereas AMP-A was almost inactive against most of the bacterial strains tested. AMP-B/C/D showed more potent activity against Gram-positive bacteria than against Gram-negative bacteria. By utilizing bioinformatics analysis tools, we found that the secondary structure of the four cation peptides was mainly alpha-helix, and the result of CD spectrum also displayed that all the peptides had considerable alpha-helix in the presence of either 50% TFE or SDS micelles. AMP-C showed much better activity than other peptides against most of the bacteria tested, owing to its remarkable cation property and the amphipathic character of its N-terminal. The study of structure-activity relationship of the designed peptides confirmed that amphipathic structure and high net positive charge were prerequisites for maintaining their activities.     

Antibacterial peptides of the moth Galleria mellonella

The work describes purification and biochemical characterization of two inducible antimicrobial peptides from the hemolymph of Galleria mellonella. The peptides were isolated by a sequence of reversed-phase chromatography steps from the hemolymph of larvae immunized with viable bacteria. The first peptide is a member of the cecropin family while the second one is rich in proline residues and has a unique sequence.     

Major royal jelly protein 2 acts as an antimicrobial agent and antioxidant in royal jelly

Royal jelly (RJ) is a well-known functional and medicinal food for human health promotion. Major royal jelly proteins (MRJPs), which are the major protein components in RJ, exhibit antimicrobial activities. However, the identities of the MRJPs of RJ responsible for its antioxidant effects have remained unclear. Here, we report that honeybee (Apis cerana) MRJP 2 (AcMRJP2) acts as an antimicrobial and antioxidant agent in RJ. Using recombinant AcMRJP2, which was produced in baculovirus-infected insect cells, we established the antimicrobial and antioxidant roles of MRJP 2. AcMRJP2 bound to the surfaces of bacteria, fungi, and yeast, which then induced structural damage in the microbial cell walls and led to a broad spectrum of antimicrobial activities. AcMRJP2 protected mammalian and insect cells via the direct shielding of the cell against oxidative stress, which led to reduced levels of caspase-3 activity and oxidative stress-induced cell apoptosis, followed by increased cell viability. Moreover, AcMRJP2 exhibited DNA protection activity against reactive oxygen species (ROS). Our data indicate that AcMRJP2 could play a crucial role as an antimicrobial agent and antioxidant in RJ, suggesting that MRJP 2 is a component responsible for the antimicrobial and antioxidant activities of RJ.     

Diversity,Antimicrobial Action and Structure-Activity Relationship of Buffalo Cathelicidins

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.     

Molecular cloning of novel antimicrobial peptide genes from the skin of the Chinese brown frog, Rana chensinensis

One species of the Chinese brown frog, Rana chensinensis, is widely distributed in north-central China. In this study, a cDNA library was constructed to clone the antimicrobial peptides' genes from the skin of R. chensinensis. Twenty-three prepropeptide cDNA sequences encoding twelve novel mature antimicrobial peptides were isolated and characterized. Six peptides belonged to three known families previously identified from other Ranid frogs: temporin (4 peptides), brevinin-2 (1 peptide), and palustrin-2 (1 peptide). The other six peptides showed little similarity to known antimicrobial peptides. According to the amino acid sequences, with or without α-helix structure, and either hydrophilic or hydrophobic, these were organized into four new families: chensinin-1 (3 peptides), chensinin-2 (1 peptide), chensinin-3 (1 peptide), and chensinin-4 (1 peptide). Five peptides from different families were chemically synthesized, and their antimicrobial, cytolytic, and hemolytic activities were evaluated. Of these, brevinin-2CE showed strongest antimicrobial activities against both the Gram-positive and Gram-negative bacteria with a slight hemolysis. Temporin-1CEe and palustrin-2CE also displayed a slight hemolysis, but they had different activities to prokaryotic cells. Temporin-1CEe showed higher antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria, whereas it was contrary to palustrin-2CE. Chensinin-1 CEb and chensinin-3CE only had moderate antimicrobial activity against microorganisms. In addition, the brevinin-2 peptides from different brown frogs were analyzed to reveal the taxonomy and phylogenetic relationships of R. chensinensis.     

Rational design of cationic antimicrobial peptides by the tandem of leucine-rich repeat

Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Lots of antimicrobial peptides were synthesized based on well-known structural motif widely existed in a variety of lives. Leucine-rich repeats (LRRs) are sequence motifs present in over 60,000 proteins identified from viruses, bacteria, and eukaryotes. To elucidate if LRR motif possesses antimicrobial potency, two peptides containing one or two LRRs were designed. The biological activity and membrane–peptide interactions of the peptides were analyzed. The results showed that the tandem of two LRRs exhibited similar antibacterial activity and significantly weaker hemolytic activity against hRBCs than the well-known membrane active peptide melittin. The peptide with one LRR was defective at antimicrobial and hemolytic activity. The peptide containing two LRRs formed α-helical structure, respectively, in the presence of membrane-mimicking environment. LRR-2 retained strong resistance to cations, heat, and some proteolytic enzymes. The blue shifts of the peptides in two lipid systems correlated positively with their biological activities. Other membrane-peptide experiments further provide the evidence that the peptide with two LRRs kills bacteria via membrane-involving mechanism. The present study increases our new understanding of well-known LRR motif in antimicrobial potency and presents a potential strategy to develop novel antibacterial agents.     

Cathelicidins: family of antimicrobial peptides. A review

Cathelicidins are small, cationic, antimicrobial peptides found in humans and other species, including farm animals (cattle, horses, pigs, sheep, goats, chickens, rabbits and in some species of fish). These proteolytically activated peptides are part of the innate immune system of many vertebrates. These peptides show a broad spectrum of antimicrobial activity against bacteria, enveloped viruses and fungi. Apart from exerting direct antimicrobial effects, cathelicidins can also trigger specific defense responses in the host. Their roles in various pathophysiological conditions have been studied in mice and humans, but there are limited information about their expression sites and activities in livestock. The aim of the present review is to summarize current information about these antimicrobial peptides in farm animals, highlighting peptide expression sites, activities, and future applications for human and veterinary medicine.