抗菌肽在宿主防御中作用

抗菌肽广泛地存在于自然界中,其中许多抗菌肽具有直接抗微生物活性,能作用于G-、 G+细菌、真菌、寄生虫甚至是包膜病毒,并且在宿主先天免疫和适应性反应中有重要的调节作用。近来,越来越多的证据表明抗菌肽是有效的免疫辅助因子,能够与其他的众多免疫效应子协同作用,从而起始适应性免疫,促进伤口愈合,抑制前炎症反应以及诱导和调节细胞因子和趋化因子的产生。另外,随着抗菌肽作用机理逐渐被揭开,将这些内源性肽及其衍生物制成抗感染治疗药剂将会有广阔的应用前景。     

Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity

Antimicrobial host defense peptides (HDPs) are a critical component of the innate immunity with microbicidal, endotoxin-neutralizing, and immunostimulatory properties. HDPs kill bacteria primarily through non-specific membrane lysis, therefore with a less likelihood of provoking resistance. Extensive structure–activity relationship studies with a number of HDPs have revealed that net charge, amphipathicity, hydrophobicity, and structural propensity are among the most important physicochemical and structural parameters that dictate their ability to interact with and disrupt membranes. A delicate balance among these factors, rather than a mere alteration of a single factor, is critically important for HDPs to ensure the antimicrobial potency and target cell selectivity. With a better understanding of the structural determinants of HDPs for their membrane-lytic activities, it is expected that novel HDP-based antimicrobials with minimum toxicity to eukaryotic cells can be developed for resistant infections, which have become a global public health crisis.     

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

Antimicrobial peptides (AMPs), with their extraordinary properties, such as broad-spectrum activity, rapid action and difficult development of resistance, have become promising molecules as new antibiotics. Despite their various mechanisms of action, the interaction of AMPs with the bacterial cell membrane is the key step for their mode of action. Moreover, it is generally accepted that the membrane is the primary target of most AMPs, and the interaction between AMPs and eukaryotic cell membranes (causing toxicity to host cells) limits their clinical application. Therefore, researchers are engaged in reforming or de novo designing AMPs as a ‘single-edged sword’ that contains high antimicrobial activity yet low cytotoxicity against eukaryotic cells. To improve the antimicrobial activity of AMPs, the relationship between the structure and function of AMPs has been rigorously pursued. In this review, we focus on the current knowledge of α-helical cationic antimicrobial peptides, one of the most common types of AMPs in nature.     

Bacterial resistance to antimicrobial peptides

Antimicrobial peptides (AMPs) or host defense peptides (HDPs) are vital components of human innate defense system targeting human‐related bacteria. Many bacteria have various mechanisms interfering with AMP activity, causing resistance to AMPs. Since AMPs are considered as potential novel antimicrobial drugs, understanding the mechanisms of bacterial resistance to direct killing of AMPs is of great significance. In this review, a comparative overview of bacterial strategies for resistance to direct killing of various AMPs is presented. Such strategies include bacterial cell envelope modification, AMP degradation, sequestration, expelling, and capsule.     

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.     

Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

Artificial peptides to induce membrane denaturation and disruption and modulate membrane composition and fusion

Membranes consisting of phospholipid bilayers are an essential constituent of eukaryotic cells and their compartments. The alteration of their composition, structure, and morphology plays an important role in modulating physiological processes, such as transport of molecules, cell migration, or signaling, but it can also lead to lethal effects. The three main classes of membrane-active peptides that are responsible for inducing such alterations are cell-penetrating peptides (CPPs), antimicrobial peptides (AMPs), and fusion peptides (FPs). These peptides are able to interact with lipid bilayers in highly specific and tightly regulated manners. They can either penetrate the membrane, inducing nondestructive, transient alterations, or disrupt, permeabilize, or translocate through it, or induce membrane fusion by generating attractive forces between two bilayers. Because of these properties, membrane-active peptides have attracted the attention of the pharmaceutical industry, and naturally occurring bioactive structures have been used as a platform for synthetic modification and the development of artificial analogs with optimized therapeutic properties to transport biologically active cargos or serve as novel antimicrobial agents. In this review, we focus on synthetic membrane interacting peptides with bioactivity comparable with their natural counterparts and describe their mechanism of action.     

The heme sensing response regulator HssR in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> but not the homologous RR23 in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> modulates susceptibility to the antimicrobial peptide plectasin

Background  

Host defence peptides (HDPs), also known as antimicrobial peptides (AMPs), have emerged as potential new therapeutics and their antimicrobial spectrum covers a wide range of target organisms. However, the mode of action and the genetics behind the bacterial response to HDPs is incompletely understood and such knowledge is required to evaluate their potential as antimicrobial therapeutics. Plectasin is a recently discovered HDP active against Gram-positive bacteria with the human pathogen, Staphylococcus aureus (S. aureus) being highly susceptible and the food borne pathogen, Listeria monocytogenes (L. monocytogenes) being less sensitive. In the present study we aimed to use transposon mutagenesis to determine the genetic basis for S. aureus and L. monocytogenes susceptibility to plectasin.     

Antimicrobial peptides effectively kill a broad spectrum of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains independently of origin,sub-type,or virulence factor expression

Background  

Host defense peptides (HDPs), or antimicrobial peptides (AMPs), are important components of the innate immune system that bacterial pathogens must overcome to establish an infection and HDPs have been suggested as novel antimicrobial therapeutics in treatment of infectious diseases. Hence it is important to determine the natural variation in susceptibility to HDPs to ensure a successful use in clinical treatment regimes.     

Mechanical properties that influence antimicrobial peptide activity in lipid membranes

Antimicrobial peptides are small amphiphilic proteins found in animals and plants as essential components of the innate immune system and whose function is to control bacterial infectious activity. In order to accomplish their function, antimicrobial peptides use different mechanisms of action which have been deeply studied in view of their potential exploitation to treat antibiotic-resistant bacterial infections. One of the main mechanisms of action of these peptides is the disruption of the bacterial membrane through pore formation, which, in some cases, takes place via a monomer to oligomer cooperative transition. Previous studies have shown that lipid composition, and the presence of exogenous components, such as cholesterol in model membranes or carotenoids in bacteria, can affect the potency of distinct antimicrobial peptides. At the same time, considering the membrane as a two-dimensional material, it has been shown that membrane composition defines its mechanical properties which might be relevant in many membrane-related processes. Nevertheless, the correlation between the mechanical properties of the membrane and antimicrobial peptide potency has not been considered according to the importance it deserves. The relevance of these mechanical properties in membrane deformation due to peptide insertion is reviewed here for different types of pores in order to elucidate if indeed membrane composition affects antimicrobial peptide activity by modulation of the mechanical properties of the membrane. This would also provide a better understanding of the mechanisms used by bacteria to overcome antimicrobial peptide activity.     

Machine learning-enabled discovery and design of membrane-active peptides

Antimicrobial peptides are a class of membrane-active peptides that form a critical component of innate host immunity and possess a diversity of sequence and structure. Machine learning approaches have been profitably employed to efficiently screen sequence space and guide experiment towards promising candidates with high putative activity. In this mini-review, we provide an introduction to antimicrobial peptides and summarize recent advances in machine learning-enabled antimicrobial peptide discovery and design with a focus on a recent work Lee et al. Proc. Natl. Acad. Sci. USA 2016;113(48):13588–13593. This study reports the development of a support vector machine classifier to aid in the design of membrane active peptides. We use this model to discover membrane activity as a multiplexed function in diverse peptide families and provide interpretable understanding of the physicochemical properties and mechanisms governing membrane activity. Experimental validation of the classifier reveals it to have learned membrane activity as a unifying signature of antimicrobial peptides with diverse modes of action. Some of the discriminating rules by which it performs classification are in line with existing “human learned” understanding, but it also unveils new previously unknown determinants and multidimensional couplings governing membrane activity. Integrating machine learning with targeted experimentation can guide both antimicrobial peptide discovery and design and new understanding of the properties and mechanisms underpinning their modes of action.     

Surface plasmon resonance analysis of antimicrobial peptide–membrane interactions: affinity & mechanism of action

Antimicrobial peptides are being increasingly recognised as potential candidates for antibacterial drugs in the face of the rapidly emerging bacterial resistance to conventional antibiotics in recent years. However, a precise understanding of the relationship between antimicrobial peptide structure and their cytolytic function in a range of organisms is still lacking. This is a result of the complex nature of the interactions of antimicrobial peptides with the cell membrane, the mechanism of which can vary considerably between different classes of antimicrobial peptides. A wide range of biophysical techniques have been used to study the influence of a number of peptide and membrane properties on the cytolytic activity of these peptides model membrane systems. Until recently, however, very few studies had reported measurements of the affinity of antimicrobial peptides for different membrane systems mainly due to the difficulty in obtaining this information. Surface plasmon resonance (SPR) spectroscopy has recently been applied to the study of biomembrane-based systems which has allowed a real-time analysis of binding affinity and kinetics. This mini review provides an overview of the recent applications that demonstrate the potential of SPR to study the membrane interactions of antimicrobial peptides.     

抗菌肽抗菌机制及其应用研究进展

抗菌肽是一类小分子肽,具有广谱的抗菌活性。以往对抗菌肽抗菌机制的研究主要集中在细菌细胞膜的作用上,包含"桶板"模型、"毯式"模型,"环形孔"模型和"凝聚"模型。近年来相继发现某些抗菌肽可以作用于细菌细胞内部,与核酸物质结合,阻断DNA复制、RNA合成;影响蛋白质合成;抑制隔膜、细胞壁合成,阻碍细胞分裂;抑制胞内酶的活性。本文从胞内机制和胞外机制两个角度对抗菌肽的抗菌机制进行综述,以期阐明各类抗菌肽的作用机制,为进一步研究菌株耐药性、杀菌效果及其杀菌机制提供科学根据。     

The penetrating properties of the tumor homing peptide LyP‐1 in model lipid membranes

Cell‐penetrating peptides (CPPs) have the property to cross the plasma membrane and enhance its permeability. CPPs were successfully used to deliver numerous cargoes such as drugs, proteins, nucleic acids, imaging and radiotherapeutic agents, gold and magnetic nanoparticles, or liposomes inside cells. Although CPPs were intensively investigated over the past 20 years, the exact molecular mechanisms of translocation across membranes are still controversial and vary from passive to active mechanisms. LyP‐1 is a cyclic 9‐amino‐acids homing peptide that specifically binds to p32 receptors overexpressed in tumor cells. tLyP‐1 peptide is the linear truncated form of LyP‐1 and recognizes neuropilin (NRP) receptors expressed in glioma tumor tissue. Here, we investigate the interaction of the cyclic LyP‐1 peptide and linear truncated tLyP‐1 peptide with model plasma membrane in order to understand their passive, energy‐independent mechanism of uptake. The experiments reveal that internalization of tLyP‐1 peptides depends on membrane lipid composition. Inclusion of negatively charged phosphatidylserine (PS) or cone‐shaped phosphatidylethanolamine (PE) lipids in the composition of giant unilamellar vesicles facilitates the membrane adsorption and direct penetration but without inducing pore formation in membranes. In contrast, cyclic LyP‐1 peptide mostly accumulates on the membrane, with very low internalization, regardless of the lipid composition. Thus, the linear tLyP‐1 peptide has enhanced penetrating properties compared with the cyclic LyP‐1 peptide. Development of a mutant peptide containing higher number of arginine amino acids and preserving the homing properties of tLyP‐1 may be a solution for new permeable peptides that facilitate the internalization in cells and further the endosomal escape as well.     

Preassembly of membrane-active peptides is an important factor in their selectivity toward target cells

Membrane-active peptides comprise a large group of toxins used in the defense and offense systems of all organisms including plants and humans. They act on diverse targets including microorganisms and mammalian cells, but the factors that determine their target cell selectivity are not yet clear. Here, we tested the role of peptide length and preassembly on the ability of diastereomeric cationic antimicrobial peptides to discriminate among bacteria, erythrocytes, and fungal cells, by using peptides with variable lengths (13, 16, and 19 amino acids long) and their covalently linked pentameric bundles. All the bundles expressed similar potent antifungal activity (minimal inhibitory concentration of 0.2-0.3 microM) and high antimicrobial activity. Hemolytic activity was also observed at concentrations higher than those required for antifungal activity. In contrast, all the monomers showed length-dependent antimicrobial activity, were less active toward bacteria and fungi, and were devoid of hemolytic activity. BIAcore biosensor experiments revealed a approximately 300-fold increase in peptide-membrane binding affinity between the 13- and 19-residue monomers toward zwitterionic (egg phosphatidylcholine (PC)/egg spingomyelin (SM)/cholesterol) vesicles. All the monomeric peptides display a similar high affinity to negatively charged (E. coli phosphatidylethanolamine (PE)/egg phosphatidylglycerol (PG)) vesicles regardless of their length. In contrast, irrespective of the size of the monomeric subunit, all the bundles bind irreversibly and strongly disrupt both PC/SM/cholesterol and PE/PG membranes. Attenuated total reflectance Fourier-transform infrared spectroscopy revealed that peptide assembly also affects structure as observed by an increased alpha-helical and beta-sheet content in membranes and enhances acyl chain disruption of PC/cholesterol. The correlation between the antibacterial activity and ability to depolarize the transmembrane potential of E. coli spheroplasts, as well as the ability to induce calcein release from vesicles, suggests that the bacterial membrane is their target. The data demonstrate that preassembly of cationic diastereomeric antimicrobial peptides is an essential factor in their membrane targeting.     

Phagocytosis of bacterial pathogens

Phagocytosis is an evolutionarily ancient, receptor-driven process, by which phagocytic cells recognize invading microbes and destroy them after internalization. The phagocytosis receptor Eater is expressed exclusively on Drosophila phagocytes and is required for the survival of bacterial infections. In a recent study, we explored how Eater can defend fruit flies against different kinds of bacteria. We discovered that Eater bound to certain types of bacteria directly, while for others bacterial binding was dependent on prior disruption of the bacterial envelope. Similar to phagocytes, antimicrobial peptides and lysozymes are ancient components of animal immune systems. Our results suggest that cationic antimicrobial peptides, as well as lysozymes, can facilitate Eater binding to live Gram-negative bacteria. Both types of molecules promote surface-exposure of bacterial ligands that otherwise would remain buried and hidden under an outer membrane. We propose that unmasking ligands for phagocytic receptors may be a conserved mechanism operating in many animals, including humans. Thus, studying a Drosophila phagocytosis receptor may advance our understanding of innate immunity in general.     

Phagocytosis of bacterial pathogens

Phagocytosis is an evolutionarily ancient, receptor-driven process, by which phagocytic cells recognize invading microbes and destroy them after internalization. The phagocytosis receptor Eater is expressed exclusively on Drosophila phagocytes and is required for the survival of bacterial infections. In a recent study, we explored how Eater can defend fruit flies against different kinds of bacteria. We discovered that Eater bound to certain types of bacteria directly, while for others bacterial binding was dependent on prior disruption of the bacterial envelope. Similar to phagocytes, antimicrobial peptides and lysozymes are ancient components of animal immune systems. Our results suggest that cationic antimicrobial peptides, as well as lysozymes, can facilitate Eater binding to live Gram-negative bacteria. Both types of molecules promote surface-exposure of bacterial ligands that otherwise would remain buried and hidden under an outer membrane. We propose that unmasking ligands for phagocytic receptors may be a conserved mechanism operating in many animals, including humans. Thus, studying a Drosophila phagocytosis receptor may advance our understanding of innate immunity in general.     

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.     

Natural History of Innate Host Defense Peptides

Host defense peptides act on the forefront of innate immunity, thus playing a central role in the survival of animals and plants. Despite vast morphological changes in species through evolutionary history, all animals examined to date share common features in their innate immune defense strategies, hereunder expression of host defense peptides (HDPs). Most studies on HDPs have focused on humans, domestic and laboratory animals. More than a thousand different sequences have been identified, yet data on HDPs in wild-living animals are sparse. The biological functions of HDPs include broad-spectrum antimicrobial activity and immunomodulation. Natural selection and coevolutionary host-pathogen arms race theory suggest that the extent and specificity of the microbial load influences the spectrum and potency of HDPs in different species. Individuals of extant species—that have lived for an extended period in evolutionary history amid populations with intact processes of natural selection—likely possess the most powerful and well-adapted “natural antibiotics”. Research on the evolutionary history of the innate defense system and the host in context of the consequences of challenges as well as the efficacy of the innate immune system under natural conditions is therefore of immediate interest. This review focuses on evolutionary aspects of immunophysiology, with emphasis on innate effector molecules. Studies on host defense in wild-living animals may significantly enhance our understanding of inborn immune mechanisms, and help identify molecules that may assist us to cope better with the increasing microbial challenges that likely follow from the continuous amplification of biodiversity levels on Earth.     

Chlamydia-secreted protease CPAF degrades host antimicrobial peptides

Chlamydia trachomatis infection in the lower genital tract, if untreated, can ascend to the upper genital tract, potentially leading to complications such as tubal factor infertility. The ascension involves cell-to-cell spreading, which may require C. trachomatis organisms to overcome mucosal extracellular effectors such as antimicrobial peptides. We found that among the 8 antimicrobial peptides tested, the cathelicidin LL-37 that is produced by both urogenital epithelial cells and the recruited neutrophils possessed a most potent antichlamydial activity. Interestingly, this antichlamydial activity was completely inhibited by CPAF, a C. trachomatis-secreted serine protease. The inhibition was dependent on CPAF's proteolytic activity. CPAF selectively degraded LL-37 and other antimicrobial peptides with an antichlamydial activity. CPAF is known to secrete into and accumulate in the infected host cell cytoplasm at the late stage of chlamydial intracellular growth and may be released to confront the extracellular antimicrobial peptides before the intra-inclusion organisms are exposed to extracellular environments during host cell lysis and chlamydial spreading. Thus, the finding that CPAF selectively targets host antimicrobial peptides that possess antichlamydial activities for proteolysis suggests that CPAF may contribute to C. trachomatis pathogenicity by aiding in ascending infection.