Neurogenic and septic inductions of synthesis of peptide antibiotics in larvae of <Emphasis Type="Italic">Calliphora vicina</Emphasis> R.-D. (Diptera: Calliphoridae)

Synthesis of antimicrobial peptides in diapausing larvae of the blowfly Calliphora vicina can be induced by two different pathways. One pathway is well known in insects and includes recognition of microbial particles by the pattern-recognizing receptors. The other pathway includes perception and transduction of stress signal to immunocompetent cells by neuroendocrine system. This phenomenon consists in stimulation of synthesis of defensins, cecropins, and diptericins under effect of chromic stimulation of mechanoreceptors with ligature applied on the larva head end. Formation of immune response in brain is established to need less than 30 s, after which isolation of the neuroendocrine complex does not eliminate activation of immune response. As judging from rate of the neurogenic induction, transduction of the stimulating signal from brain to the immune system cells can be connected with release into hemolymph of biogenic amines or other neurohormones stored preliminarily in the neurohemal organ. The nature of this inductor at present remains unknown, as analysis of role of octopamine, dopamine, and adipokinetic hormone did not reveal stimulating effect on synthesis of bactericidal peptides. Physiological mechanism of this phenomenon is not finally understood, its key links seem to be CNS, hormonal factor of cardiac bodies, and system of antimicrobial peptides. Synthesis of antimicrobial peptides is directly regulated by the neuroendocrine system that can produce both stimulating and stress action by reminding in this aspect the known immunoneuroendocrine interrelations in vertebrates. The existence of similar integrating mechanisms in such polar animal kingdom groups as insects and vertebrates indicate that they are more ancient than this was considered earlier.     

昆虫天然免疫反应分子机制研究进展

昆虫体内缺乏高等脊椎动物所具有的获得性免疫系统, 只能依赖发达的天然免疫系统抵抗细菌、 真菌、 病毒等外源病原物的侵染。本文概括了昆虫天然免疫反应发生和作用的分子机制相关进展, 重点阐述了重要免疫相关因子在昆虫天然免疫反应中的功能和作用机制。昆虫天然免疫反应分为体液免疫和细胞免疫两种, 二者共同作用完成对病原物的吞噬 (phagocytosis)、 集结 (nodulation)、 包囊 (encapsulation)、 凝结 (coagulation)和黑化（melanization）等。当昆虫受到外界病原物的侵染时, 首先通过体内的模式识别蛋白（pattern recognition proteins/receptor, PRPs）识别并结合病原物表面特有的模式分子（pathogen-associated molecular pattern, PAMPs）, 继而一系列包括丝氨酸蛋白酶和丝氨酸蛋白酶抑制剂在内的级联激活反应被激活和调控, 产生抗菌肽、 黑色素等免疫效应分子, 清除或杀灭外源物。抗菌肽是一类小分子量的阳离子肽, 具有广谱抗菌活性, 针对不同类型的病原物, 抗菌肽的产生机制也不尽相同。昆虫体内存在着两种信号转导途径调节抗菌肽的产生： 一是由真菌和大部分革兰氏阳性菌激活的Toll途径; 二是由革兰氏阴性菌激活的Imd途径（immune deficiency pathway）。这两个途径通过激活不同转录因子调控不同抗菌肽基因的表达参与昆虫体内的天然免疫反应。     

昆虫先天性免疫信号通路研究进展

昆虫体内形成了强大的免疫防御系统,其被各种微生物攻击时能依靠病原相关分子模式识别蛋白对感染进行区分和激活体内信号通路诱导如抗菌肽之类的效应分子.昆虫体内控制先天性免疫的信号通路分别是:Toll通路、IMD通路和JAS/STAT通路,这3条通路在信号传递过程中存在协作,并且,这些通路与脊椎动物体内某些通路存在惊人相似、在免疫调控通路方面存在共同的进化起源.这揭示了先天性免疫在动物体内存在的普遍性和机体抵御病原感染的重要性.     

果蝇抗微生物肽基因的免疫诱导模式

果蝇作为一种模式昆虫,为研究昆虫和人类的先天免疫发挥了重要作用。目前对果蝇体内免疫诱导产生的抗微生物肽多基因家族在分子进化、抗菌功能的分子特征和免疫诱导表达的信号传递机制等方面的研究进展,进一步加深了人们对昆虫乃至其他动物和人类先天免疫模式的认识,为研究其他昆虫特别是作为主要农林害虫的鳞翅目昆虫的先天免疫机制发挥了重要作用。本文集中对黑腹果蝇Drosophila melanogaster抗微生物肽及其免疫模式的研究结果和最新进展进行了介绍,其中包括作者近几年的研究结果。     

昆虫天然免疫相关基因研究进展

在长期进化过程中,昆虫形成了强大的天然免疫防御系统,即体液免疫和细胞免疫。体液免疫主要包括Toll、IMD和JAK/STAT 3条信号通路,通过信号转导及免疫途径调控免疫相关基因的表达,诱导产生抗菌肽和其他效应分子。细胞免疫由血细胞介导,主要完成对病原物的包裹、吞噬和集结等。近年来,昆虫基因组学快速发展,通过生物信息学等方法从昆虫基因组数据中已鉴定到大量免疫相关基因,对这些基因的研究加深了人们对昆虫天然免疫分子机制的认识和理解。根据基因功能,免疫相关基因分为识别、信号转导、调制器、效应分子、黑化反应、RNA干扰和其他基因等7类,这些基因通过互作来调控体液免疫和细胞免疫。本文对昆虫免疫相关基因的分类、功能及家族进化等方面的研究成果进行总结,并对今后昆虫免疫的研究重点进行了展望,以期为昆虫免疫分子机制的研究及开发新的害虫防治策略提供依据。     

Induction of antimicrobial peptide synthesis by the fat body cells of maggots of <Emphasis Type="Italic">Calliphora vicina</Emphasis> R.-D. (Diptera,Calliphoridae)

Fat body is the major site of peptide antibiotic synthesis in insects. The goal of this work was to search in Calliphora vicina maggot for humoral factors that induce synthesis of antimicrobial peptides by the fat body cells in trauma. The preliminary analysis has shown that the activation factor revealed in hemolymph is a thermostable hydrophilic compound with a molecular mass lower than 3 kDa. The integument epitheliocytes were also found to release the humoral factors directly stimulating synthesis of antimicrobial peptides by the fat body.     

泛素-蛋白酶体系统在昆虫先天免疫中的调控作用

完善的先天免疫系统使得昆虫成为分布最广、适应性最强、物种多样性最丰富的动物类群。在长期的进化过程中,昆虫建立了一套安全有效的先天免疫系统,一方面在面对外界微生物攻击的时候及时有效的发生免疫应答反应;另一方面通过免疫抑制来调控适度免疫应答,避免对自身发动攻击和控制环境共生菌刺激引起的免疫应答信号通路的持续激活。泛素-蛋白酶体系统在昆虫先天免疫中具有重要的调控作用,在Toll和IMD信号通路中,通过对免疫应答通路中信号分子的泛素化修饰加工,促进或抑制抗菌肽的表达,从而使免疫反应达到一个平衡。本文通过对泛素-蛋白酶体系统在Toll和IMD信号通路中的免疫应答和免疫抑制方面的研究进行综述,阐明了该系统在昆虫先天免疫中的调控作用,将有助于开展农业害虫与其天敌之间相互关系的深入研究,揭示其免疫调控机理,为开发生物农药,进行生物防控提供理论依据。     

Opioid mechanisms in insects,with special attention toLeucophaea maderae

1. This review article provides information on the evolutionary history of neuroendocrine and related regulatory mechanisms. It focuses on the presence, diverse roles, and modes of operation of one class of neuropeptides, the endogenous opioids, in insects. 2. Opioid peptides, closely resembling those of vertebrates, have been identified in the brain and related neuroendocrine structures by means of immunocytochemistry and high-pressure liquid chromatography. 3. The demonstration of naloxone-sensitive, high-affinity binding sites for Met-enkephalin-like neuropeptides in the brain and digestive tract of Leucophaea deserves special attention because it provides new insights into the functional significance of opiate receptors paralleling those known in vertebrates. 4. Possible roles of receptor-mediated opioid systems in the insects discussed are regulation of the cyclicity of the female reproductive system, maintenance of normal midgut function mediated by the recurrent nerve, and locomotor activity.     

Induction of antimicrobial peptide synthesis by fat body cells in Calliphora vicina R.-D. (diptera, calliphoridae) larvae

The fat body is the main place of synthesis if peptide antibiotics in insects. The goal of the present study was the search in the body of the larva Calliphora vicina for humoral factors inducing synthesis of antimicrobial peptides by fat body cells during trauma. The preliminary analysis has shown that the activation factor revealed in hemolymph is the thermostable hydrophilic compound with molecular mass less than 3 kDa. According to results of experiments, the integument epitheliocytes and hemocytes also release the humoral factors directly stimulating synthesis of antimicrobial peptides by fat body.     

Cytotoxicity and cytotoxic molecules in invertebrates

Although lacking the components that characterize the acquired immunity systems of vertebrates, invertebrates nevertheless possess effective general innate immune mechanisms which exhibit striking parallels with those of vertebrates. These innate immune systems include both cellular and humoral elements. Invertebrate phagocytes synthesize both oxygen-dependent and oxygen-independent molecules to combat infectious agents. Cytotoxic substances employed by invertebrates include reactive intermediates of oxygen and nitrogen, antimicrobial peptides, lectins, cytokine- and complement-like molecules, and quinoid intermediates of melanin. The signal transduction pathways that are involved in mediating the production of these substances appear to be very similar among animal species, suggesting a common ancestral origin for the innate immune systems.     

FADD形成淀粉样蛋白纤维及其在果蝇免疫信号传递中的作用

探究果蝇FADD(Fas-associateddeathdomain-containingprotein)淀粉样蛋白纤维的形成及对IMD(Immune deficiency)信号通路中信号传递的影响,有助于更清楚地了解昆虫先天免疫信号通路中的调节机制,为其他物种的免疫调控提供参考。通过原核表达纯化dFADD蛋白、硫黄素T结合和透射电子显微镜观察等鉴定dFADD在体外纤维的形成;通过构建dFADD真核表达载体,SDD-AGE检测、共聚焦显微镜观察等探究dFADD在果蝇S2细胞内纤维聚合物的形成;构建dFADD结构域突变体,检测纤维形成的关键结构域及对IMD信号传递的影响。结果表明,dFADD在体外和细胞水平上都能聚合形成淀粉样蛋白纤维聚合物;纤维的形成是由dFADD的DED (Death-effector domain)结构域决定的,当DED结构域缺失时,dFADD以单体形式存在;双荧光素酶报告系统的检测结果显示,dFADD只有形成纤维时,才能诱导下游抗菌肽的表达,表明纤维形成是IMD信号传递的关键。本研究揭示了dFADD通过形成淀粉样蛋白纤维参与IMD信号通路中介导IMD与Dredd级联传导的作用,进一步加深了对淀粉样蛋白纤维不仅在哺乳动物,也在昆虫的免疫信号通路中传递信号这一保守功能的认识。     

Fungal peptide Destruxin A plays a specific role in suppressing the innate immune response in Drosophila melanogaster

Destruxins are a class of insecticidal, anti-viral, and phytotoxic cyclic depsipeptides that are also studied for their toxicity to cancer cells. They are produced by various fungi, and a direct relationship has been established between Destruxin production and the virulence of the entomopathogen Metarhizium anisopliae. Aside from opening calcium channels, their in vivo mode of action during pathogenesis remains largely uncharacterized. To better understand the effects of a Destruxin, we looked at changes in gene expression following injection of Destruxin A into the fruit fly Drosophila melanogaster. Microarray results revealed reduced expression of various antimicrobial peptides that play a major role in the humoral immune response of the fly. Flies co-injected with a non-lethal dose of Destruxin A and the normally innocuous Gram-negative bacteria Escherichia coli, showed increased mortality and an accompanying increase in bacterial titers. Mortality due to sepsis was rescued through ectopic activation of components in the IMD pathway, one of two signal transduction pathways that are responsible for antimicrobial peptide induction. These results demonstrate a novel role for Destruxin A in specific suppression of the humoral immune response in insects.     

果蝇先天性免疫研究进展

果蝇是生命科学与人类疾病研究的重要模式生物,虽然不具有人类高度专一的获得性免疫,但也有对病原微生物感染作出快速有效反应的先天性免疫应答系统,主要包括体液免疫,细胞免疫和黑化反应。文章结合国外最新研究,详细介绍果蝇体液免疫中控制抗菌肽合成的Toll信号通路和Imd信号通路中涉及的蛋白及其相互作用,并对果蝇细胞免疫中的吞噬、包埋功能和黑化反应作简要阐述。研究表明,果蝇的Toll和Imd信号通路分别与人类的TLR4和TNRF-1信号通路存在着惊人的相似之处,说明果蝇与人类在免疫调控通路方面可能存在着共同的进化起源。     

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems.     

Novel properties of antimicrobial peptides

Endogenous peptide antibiotics are known as evolutionarily old components of innate immunity. Due to interaction with cell membrane these peptides cause permeabilization of the membrane and lysis of invading microbes. However, some studies proved that antimicrobial peptides are universal multifunctional molecules and their functions extend far beyond simple antibiotics. In this review we present an overview of the general mechanism of action of antimicrobial peptides and discuss some of their additional properties, like antitumour activity, mitogenic activity, role in signal transduction pathways and adaptive immune response.     

蜜蜂抗菌肽研究及其应用进展

抗菌肽是一类由特定基因编码的小分子多肽,广泛分布于各种生物中,是生物天然免疫的重要效应分子,其对缺乏获得性免疫系统的昆虫尤为重要。蜜蜂是一种对环境极其重要的社会性模式昆虫,又有着极高的经济价值,因此蜜蜂抗菌肽有着较大的研究意义。本文对蜜蜂4种天然免疫抗菌肽(Apidaecin、Abaecin、Hymenoptaecin和Defensin)和蜂产品中的抗菌肽(Jelleines、Melittin和Apamin)研究进展进行了综述,介绍了它们的功能、作用机制及其应用,提出了蜜蜂抗菌肽未来可行的研究方向,旨在推动蜜蜂抗菌肽的研究。     

Proline-rich antimicrobial peptides: potential therapeutics against antibiotic-resistant bacteria

The increasing resistance of pathogens to antibiotics causes a huge clinical burden that places great demands on academic researchers and the pharmaceutical industry for resolution. Antimicrobial peptides, part of native host defense, have emerged as novel potential antibiotic alternatives. Among the different classes of antimicrobial peptides, proline-rich antimicrobial peptides, predominantly sourced from insects, have been extensively investigated to study their specific modes of action. In this review, we focus on recent developments in these peptides. They show a variety of modes of actions, including mechanism shift at high concentration, non-lytic mechanisms, as well as possessing different intracellular targets and lipopolysaccharide binding activity. Furthermore, proline-rich antimicrobial peptides display the ability to not only modulate the immune system via cytokine activity or angiogenesis but also possess properties of penetrating cell membranes and crossing the blood brain barrier suggesting a role as potential novel carriers. Ongoing studies of these peptides will likely lead to the development of more potent antimicrobial peptides that may serve as important additions to the armoury of agents against bacterial infection and drug delivery.