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。     

抗菌肽作用机制的研究进展

抗菌肽是一类来源于多种生物、能有效杀灭病原体的小分子多肽,具有活性谱广、作用强且迅速、不易产生耐药等众多优点.作为新一代抗感染候选药物,抗菌肽的作用机制还未完全清楚,但目前有两种观点已得到公认,即胞膜渗透作用破坏胞膜结构完整性和作用于胞内不同靶点干扰细菌生长及代谢平衡.本文主要就抗菌肽理化性质、二级结构、作用机制以及后两者间的关系做一总结,以便更好的理解抗菌肽的构效关系,为合理设计抗菌肽提供理论基础.     

Surface-active antifungal polyquaternary amine

There is a distinct need for antimicrobial compounds that can act at surfaces without leaching into the environment. Such materials should be easy to synthesize, be easy to apply to surfaces, and display reasonable levels of antimicrobial and antifungal activity. Here we describe such a surface-active compound and demonstrate its ability to inhibit the growth of the filamentous fungus Aspergillus niger. We have synthesized a series of polyquaternary ammonium compounds by atom transfer radical polymerization. Two members of this series were tested for their ability to inhibit the growth of A. niger. The compounds were dried onto surfaces, and the treated surfaces were then used as growth chambers for A. niger. A water soluble polyquaternary amine compound was shown to effectively kill A. niger in solution in a dose-dependent manner. Conversely, a water insoluble polyquaternary amine compound was shown to kill only the fungi in direct contact with the material on the surface. These results have important implications for the development of effective, environmentally benign, surface-active anti-fungal compounds.     

Neutrophil extracellular traps: casting the NET over pathogenesis

Neutrophil extracellular traps (NETs) are considered to be part of the human innate immunity because they trap and kill pathogens. NETs are formed by activated neutrophils and consist of a DNA backbone with embedded antimicrobial peptides and enzymes. They are involved in host defense during pneumococcal pneumonia, streptococcal necrotizing fasciitis, appendicitis and insemination. Recently, bacterial virulence factors that counteract NETs have been identified. These include the degradation of the NET-backbone by DNases enabling the liberation of bacteria from NETs, as well as capsule formation, which reduces bacterial trapping. Furthermore, pathogens can resist NET-mediated killing by adding positive charge to their cell surface.     

The role of nitric oxide in lung innate immunity: Modulation by surfactant protein-A

Surfactant protein A (SP-A) and alveolar macrophages are essential components of lung innate immunity. Alveolar macrophages phagocytose and kill pathogens by the production of reactive oxygen and nitrogen species. In particular, peroxynitrite, the reaction product of superoxide and nitric oxide, appears to have potent antimicrobial effects. SP-A stimulates alveolar macrophages to phagocytose and kill pathogens and is important in host defense. However, SP-A has diverse effects on both innate and adaptive immunity, and may stimulate or inhibit immune function. SP-A appears to mediate toxic or protective effects depending on the immune status of the lung. In contrast to mouse or rat cells, it has been difficult to demonstrate nitric oxide production by human macrophages. We have recently demonstrated that human macrophages produce nitric oxide and use it to kill Klebsiella pneumoniae. SP-A either stimulates or inhibits this process, depending on the activation state of the macrophage. Given its diverse effects on immune function, SP-A may prove to be an effective therapy for both infectious and inflammatory diseases of the lung.     

The role of nitric oxide in lung innate immunity: modulation by surfactant protein-A

Surfactant protein A (SP-A) and alveolar macrophages are essential components of lung innate immunity. Alveolar macrophages phagocytose and kill pathogens by the production of reactive oxygen and nitrogen species. In particular, peroxynitrite, the reaction product of superoxide and nitric oxide, appears to have potent antimicrobial effects. SP-A stimulates alveolar macrophages to phagocytose and kill pathogens and is important in host defense. However, SP-A has diverse effects on both innate and adaptive immunity, and may stimulate or inhibit immune function. SP-A appears to mediate toxic or protective effects depending on the immune status of the lung. In contrast to mouse or rat cells, it has been difficult to demonstrate nitric oxide production by human macrophages. We have recently demonstrated that human macrophages produce nitric oxide and use it to kill Klebsiella pneumoniae. SP-A either stimulates or inhibits this process, depending on the activation state of the macrophage. Given its diverse effects on immune function, SP-A may prove to be an effective therapy for both infectious and inflammatory diseases of the lung.     

Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense

The production of antimicrobial peptides and proteins is essential for defense against infection. Many of the known human antimicrobial peptides are multifunctional, with stimulatory activities such as chemotaxis while simultaneously acting as natural antibiotics. In humans, eccrine appendages express DCD and CAMP, genes encoding proteins processed into the antimicrobial peptides dermcidin and LL-37. In this study we show that after secretion onto the skin surface, the CAMP gene product is processed by a serine protease-dependent mechanism into multiple novel antimicrobial peptides distinct from the cathelicidin LL-37. These peptides show enhanced antimicrobial action, acquiring the ability to kill skin pathogens such as Staphylococcus aureus and Candida albicans. Furthermore, although LL-37 may influence the host inflammatory response by stimulating IL-8 release from keratinocytes, this activity is lost in subsequently processed peptides. Thus, a single gene product encoding an important defense molecule alters structure and function in the topical environment to shift the balance of activity toward direct inhibition of microbial colonization.     

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.     

Polypropylene with embedded copper metal or copper oxide nanoparticles as a novel plastic antimicrobial agent

Aims: To develop novel polypropylene composite materials with antimicrobial activity by adding different types of copper nanoparticles. Methods and Results: Copper metal (CuP) and copper oxide nanoparticles (CuOP) were embedded in a polypropylene (PP) matrix. These composites present strong antimicrobial behaviour against E. coli that depends on the contact time between the sample and the bacteria. After just 4 h of contact, these samples are able to kill more than 95% of the bacteria. CuOP fillers are much more effective eliminating bacteria than CuP fillers, showing that the antimicrobial property further depends on the type of copper particle. Cu²⁺ released from the bulk of the composite is responsible for this behaviour. Moreover, PP/CuOP composites present a higher release rate than PP/CuP composites in a short time, explaining the antimicrobial tendency. Conclusions: Polypropylene composites based on copper nanoparticles can kill E. coli bacteria depending on the release rate of Cu²⁺ from the bulk of the material. CuOP are more effective as antimicrobial filler than CuP. Significance and Impact of the Study: Our findings open up novel applications of these ion‐copper‐delivery plastic materials based on PP with embedded copper nanoparticles with great potential as antimicrobial agents.     

中性粒细胞胞外诱捕网在炎症相关疾病中的研究进展

中性粒细胞是抵御病原体入侵机体的第一道防线,通过趋化和吞噬作用使病原体失活,从而进行免疫防御,杀灭病原体。研究证实,中性粒细胞通过吞噬病原体、分泌抗微生物蛋白颗粒来杀灭病原微生物。2004年Brinkmann发现了一种中性粒细胞新型抗感染机制,即中性粒细胞经病原体活化刺激后释放中性粒细胞胞外诱捕网(neutrophil extracellular trap,NET)至细胞外。NET是由双链DNA染色质和镶嵌在染色质上的抗菌蛋白构成的纤维网格状结构,通过网罗、捕获而杀灭病原体。诸多研究表明,NET在炎症相关疾病中起重要作用,其生成和降解会影响急慢性炎性疾病的病理过程。本文主要从NET的特征、产生机制、抗菌作用及其在炎性相关疾病中的作用等方面着手,概述其最新研究进展,为炎性疾病的治疗及其药物开发提供新的思路和方向。     

Immunomodulators as an antimicrobial tool

The spectrum of infectious diseases has shifted in the past 50 years to include those caused by microbes that cause disease predominantly in immunocompromised individuals. This phenomenon has underscored the dependence of microbial virulence on the immune status of the host. The limited efficacy of the available antimicrobial armamentarium in immunocompromised individuals, combined with increasing resistance to these agents, has led to an urgent need for new therapies for infectious diseases. Immunomodulation represents a novel approach to antimicrobial therapy that depends on bolstering host immunity, rather than direct antimicrobial activity. Immunomodulators can be divided into those that are specific to pathogens (pathogen-specific) and those that are not specific to pathogens (non-specific). However, to date only a few immunomodulators have been evaluated for their efficacy as antimicrobial tools.     

鱼精蛋白抑菌机理及在食品防腐中的应用

鱼精蛋白是一类天然的阳离子抗菌肽,具有广谱抑菌活性。鱼精蛋白主要是通过破坏细菌的细胞壁、细胞膜及改变细胞的渗透性等途径抑制甚至杀死细菌细胞。在鱼精蛋白抑制细菌的同时,细菌也产生多种机制对抗鱼精蛋白。温度、pH、阳离子和EDTA等多种理化因子影响鱼精蛋白对细菌的抑制效果。由于鱼精蛋白在抑菌防腐方面的众多优势,目前已成为非常有发展前景的食品防腐剂。     

An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps

Streptococcus pneumoniae (pneumococcus) is the most common cause of community-acquired pneumonia, with high morbidity and mortality worldwide. A major feature of pneumococcal pneumonia is an abundant neutrophil infiltration . It was recently shown that activated neutrophils release neutrophil extracellular traps (NETs), which contain antimicrobial proteins bound to a DNA scaffold. NETs provide a high local concentration of antimicrobial components and bind, disarm, and kill microbes extracellularly. Here, we show that pneumococci are trapped but, unlike many other pathogens, not killed by NETs. NET trapping in the lungs, however, may allow the host to confine the infection, reducing the likelihood for the pathogen to spread into the bloodstream. DNases are expressed by many Gram-positive bacterial pathogens, but their role in virulence is not clear. Expression of a surface endonuclease encoded by endA is a common feature of many pneumococcal strains. We show that EndA allows pneumococci to degrade the DNA scaffold of NETs and escape. Furthermore, we demonstrate that escaping NETs promotes spreading of pneumococci from the upper airways to the lungs and from the lungs into the bloodstream during pneumonia.     

Neutrophil cell signaling in infection: role of phosphatidylinositide 3-kinase

Neutrophils play a pivotal role in the innate immune response to microbial pathogens. They are uniquely suited to this role by virtue of specialized antimicrobial capabilities that include the capacity to sense minute amounts of microbial products and inflammatory mediators, to move to the site of infection, and finally to bind, internalize and kill the pathogens. To optimize host defense capabilities while minimizing damage to host tissues ('collateral damage'), these microbicidal responses must be tightly regulated. Additionally, neutrophils clear inflammatory debris, a process that is necessary for restoration of the native architecture and function of the tissue. This review highlights some recent advances in our knowledge of cell signaling as it pertains to neutrophil function, with specific emphasis on the role of the phosphatidylinositide 3-kinase in antimicrobial function.     

The bactericidal/permeability-increasing protein (BPI) in the innate defence of the lower airways

The human BPI (bactericidal/permeability-increasing protein), stored in primary azurophilic granula of neutrophil granulocytes and produced by mucosal epithelia, has been known for decades to bind LPS (lipopolysaccharide) with very high affinity and to efficiently kill Gram-negative bacteria. Thus BPI potentially represents a central component of the innate immune system to directly combat microbes and modulate subsequent adaptive immune responses. Especially in the lungs, which are frequently exposed to a variety of inhaled pathogens, antimicrobial innate defence molecules such as BPI, are of exceptional relevance. In the present review, we highlight possible functions of BPI during acute pneumonia and CF (cystic fibrosis)-associated chronic infections in the lung.     

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria¹, fungi² and parasites³. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent⁴. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase⁵. Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility⁶.We describe methods to isolate Neutrophil Granulocytes from peripheral human blood⁷ and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.     

Techno-economic analysis of a plant-based platform for manufacturing antimicrobial proteins for food safety

Continuous reports of foodborne illnesses worldwide and the prevalence of antibiotic-resistant bacteria mandate novel interventions to assure the safety of our food. Treatment of a variety of foods with bacteriophage-derived lysins and bacteriocin-class antimicrobial proteins has been shown to protect against high-risk pathogens at multiple intervention points along the food supply chain. The most significant barrier to the adoption of antimicrobial proteins as a food safety intervention by the food industry is the high production cost using current fermentation-based approaches. Recently, plants have been shown to produce antimicrobial proteins with accumulation as high as 3 g/kg fresh weight and with demonstrated activity against major foodborne pathogens. To investigate potential economic advantages and scalability of this novel platform, we evaluated a highly efficient transgenic plant-based production process. A detailed process simulation model was developed to help identify economic “hot spots” for research and development focus including process operating parameters, unit operations, consumables, and/or raw materials that have the most significant impact on production costs. Our analyses indicate that the unit production cost of antimicrobial proteins in plants at commercial scale for three scenarios is $3.00–6.88/g, which can support a competitive selling price to traditional food safety treatments.     

A microcalorimetric comparison of the anti-Streptococcus mutans efficacy of plant extracts and antimicrobial agents in oral hygiene formulations

AIMS: This study aimed to evaluate the efficacy of 'natural' putative antimicrobial agents against Streptococcus mutans and to compare these with synthetic agents using the flow microcalorimeter. Streptococcus mutans is one of the oral pathogens responsible for dental caries. METHODS AND RESULTS: Traditional microbiological techniques are invasive and destructive unlike flow microcalorimetry. This rapid technique was used to continuously monitor the power output (bioactivity) of Strep. mutans with reproducibility, precision and accuracy. The antibacterial agents found in oral hygiene products and all the natural agents tested showed anti-Strep. mutans ability. CONCLUSION: In this study microcalorimetry identified agents that had a biological effect and quantified the rate of kill achieved enabling four broad categories of antimicrobial agent to be defined. SIGNIFICANCE AND IMPACT OF THE STUDY: Microcalorimetric data are a better indication of antimicrobial efficacy than merely determining concentrations at which an antimicrobial agent is bacteriostatic or bactericidal.     

Antimicrobial and antibiofilm activity of polyurethane/Hypericum perforatum extract (PHPE) composite

Microbial accumulation in materials used in sectors such as medical, textile and food can lead to serious diseases, infections and uncontrollable problems. Many of the materials used in the above-mentioned industries have highly sensitive surfaces for microorganisms and cause colonization and biofilm formation. Colonization and biofilm formation threaten human health and they cause many diseases that result in death every year. Antimicrobial materials have an important role in combating pathogens. This article is about a new material with antibiofilm and antimicrobial properties combining polyurethane and Hypericum perforatum extract (PHPE) together. Antimicrobial effect of H. perforatum extract was determined against three clinical pathogens; C. albicans, E. coli and S. aureus. The highest antimicrobial activity of H. perforatum extract was found against S. aureus strain. Antibiofilm analysis results revealed that H. perforatum was also inhibited by the biofilm formation of S. aureus by 56.85%. The combination of polyurethane material and H. perforatum extract (PHPE) resulted in 92.85% decrease in S. aureus biofilm compared to control group. The reduction of S. aureus after H. perforatum incorporation was revealed by Scanning Electron Microscopy (SEM) study. The results show that the polyurethane material combined with H. perforatum extract inhibits the formation of S. aureus biofilm.     

A colorimetric assay for rapid screening of antimicrobial peptides

The increased resistance of various bacteria toward available antibiotic drugs has initiated intensive research efforts into identifying new sources of antimicrobial substances. Short antibiotic peptides (10-30 residues) are prevalent in nature as part of the intrinsic defense mechanisms of most organisms and have been proposed as a blueprint for the design of novel antimicrobial agents. Antimicrobial peptides are generally believed to kill bacteria through membrane permeabilization and extensive pore-formation. Assays providing rapid and easy evaluation of interactions between antimicrobial membrane peptides and lipid bilayers could significantly improve screening for substances with effective antibacterial properties, as well as contribute to the elucidation of structural and functional properties of antimicrobial peptides. Here we describe a colorimetric sensor in which particles composed of phospholipids and polymerized polydiacetylene (PDA) lipids were shown to exhibit striking color changes upon interactions with antimicrobial membrane peptides. The color changes in the system occur because of the structural perturbation of the lipids following their interactions with antimicrobial peptides. The assay was also sensitive to the antibacterial properties of structurally and functionally related peptide analogs.