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.     

Avian antimicrobial peptides: the defense role of beta-defensins

Avian antimicrobial peptides, classified as beta-defensins, have been identified from bloods of chicken, turkey, and ostrich; epithelial cells of chicken and turkey; and king penguin stomach contents. Beta-defensins are a family of antimicrobial peptides characterized by six cysteine residues forming beta-defensin motifs that are also found in bovine, ovine, pig, and human. These peptides are active against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and yeast. Analysis of evolutionary relationships of vertebrate beta-defensins showed that there might be a common ancestral gene between avian and other mammalian peptides. This ancient gene may have been passed down and evolved from species older than the oldest living birds, forming a beta-defensin-like precursor molecule. This review describes potential applications of these peptides in health care products.     

宿主防御肽抗病毒感染机制研究进展

宿主防御肽是一类广泛存在于脊椎动物的小分子多肽,具有广谱的抗菌活性以及抗炎、细胞趋化、促进血管生成和修复损伤等免疫调节功能。以往的研究多集中在宿主防御肽抗细菌和真菌感染的研究上。近年来大量研究发现,宿主防御肽也具有广泛的抗病毒活性,在临床各类病毒病的预防和治疗上具有潜在的应用前景。本文围绕宿主防御肽直接杀伤病毒、调节病毒感染过程和参与宿主抗病毒天然免疫调节这3个方面的作用机制进行综述,为宿主防御肽抗病毒相关研究和相关抗病毒生物药物的研发提供参考和借鉴。     

Structural congruence among membrane-active host defense polypeptides of diverse phylogeny

A requisite for efficacious host defense against pathogens and predators has prioritized evolution of effector molecules thereof. A recent multidimensional analysis of physicochemical properties revealed a novel, unifying structural signature among virtually all classes of cysteine-containing antimicrobial peptides. This motif, termed the γ-core, is seen in host defense peptides from organisms spanning more than 2.6 billion years of evolution. Interestingly, many toxins possess the γ-core signature, consistent with discoveries of their direct antimicrobial activity. Many microbicidal chemokines (kinocidins) likewise contain iterations of the γ-core motif, reconciling their antimicrobial efficacy. Importantly, these polypeptide classes have evolved to target and modulate biomembranes in protecting respective hosts against unfavorable interactions with potential pathogens or predators. Extending on this concept, the current report addresses the hypothesis that antimicrobial peptides, kinocidins, and polypeptide toxins are structurally congruent and share a remarkably close phylogenetic relationship, paralleling their roles in host-pathogen relationships. Analyses of their mature amino acid sequences demonstrated that cysteine-stabilized antimicrobial peptides, kinocidins, and toxins share ancient evolutionary relatedness stemming from early precursors of the γ-core signature. Moreover, comparative 3-D structure analysis revealed recurring iterations of antimicrobial peptide γ-core motifs within kinocidins and toxins. However, despite such congruence in γ-core motifs, the kinocidins diverged in overall homology from microbicidal peptides or toxins. These findings are consistent with observations that chemokines are not toxic to mammalian cells, in contrast to many antimicrobial peptides and toxins. Thus, specific functions of these molecular effectors may be governed by specific configurations of structural modules associated with a common γ-core motif. These concepts are consistent with the hypothesis that the γ-core is an archetype determinant in polypeptides that target or regulate with biological membranes, with specific iterations optimized to unique or cognate host defense contexts. Quantitative and qualitative data suggest these protein families emerged through both parallel and divergent processes of modular evolution. Taken together, the current and prior findings imply that the γ-core motif contributes to conserved structures and functions of host defense polypeptides. The presence of this unifying molecular signature in otherwise diverse categories of membrane-active host defense peptides implies an ancient and essential role for such a motif in effector molecules governing host-pathogen relationships.     

Synthetic oligoureas of metaphenylenediamine mimic host defence peptides in their antimicrobial behaviour

Oligomeric ureas of m-phenylenediamine target anionic DMPG (dimyristoylphosphatidylglycerol) and possess promise as antimicrobial agents. Their similar size, shape and hydrophobicity to helical antimicrobial peptides (AMPs) may be important for activity to exist and the ability of these compounds to insert into a well ordered lipid environment.     

Antimicrobial peptides are present in immune and host defense cells of the human respiratory and gastroinstestinal tracts

Previous studies have implicated antimicrobial peptides in the host defense of the mammalian intestinal and respiratory tract. The aim of the present study has been to characterize further the expression of these molecules in non-epithelial cells of the human pulmonary and digestive systems by detailed immunohistochemical analysis of the small and large bowel and of the large airways and lung parenchyma. Additionally, cells obtained from bronchoalveolar lavage were analyzed by fluorescent activated cell sorting and immunostaining of cytospin preparations. hBD-1, hBD-2, and LL-37 were detected in lymphocytes and macrophages in the large airways, lung parenchyma, duodenum, and colon. Lymphocytes positive for the peptides revealed a staining pattern and distribution that largely matched that of CD3-positive and CD8-positive T-cells. Macrophages with positive staining for the antimicrobial peptides also stained positively for CD68 and CD74. In view of the morphology of the LL-37-positive and hBD-2-positive mucosal lymphocytes, they are probably also B-cells. Thus, antimicrobial peptides of the defensin and cathelicidin families are present in a variety of non-epithelial cells of mucosal organs. These findings confirm that antimicrobial peptides have multiple functions in the biology of the mucosa of these organs. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ba 1641/5–1 and Ba 1641/6–1)     

Correlation between simulated physicochemical properties and hemolycity of protegrin-like antimicrobial peptides: predicting experimental toxicity

The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are beta-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five protegrin analogues newly synthesized and tested for toxicity.     

Action mechanism and structural requirements of the antimicrobial peptides, gaegurins

Gaegurins (GGNs) are a family of cationic, α-helical, antimicrobial peptides that were isolated from a Korean frog, Glandirana emeljanovi (formerly classified as Rana rugosa) and represent one of the structurally well-characterized groups. Among six gaegurins, gaegurin 4 (renamed herein esculentin-2EM), gaegurin 5 (brevinin-1EMa), and gaegurin 6 (brevinin-1EMb) have been investigated comprehensively in terms of structure-activity relationships. In this paper, we first suggest renaming of gaegurins according to a recently raised rule of systematic nomenclature. Then, the current understanding of gaegurins is reviewed by summarizing their structure-activity relationships. In particular competing arguments on gaegurins are synthetically inspected. Finally their action mechanism and structural requirements will be discussed.     

Antimicrobial and immunomodulatory activity of host defense peptides,clavanins and LL-37, in vitro: An endodontic perspective

Endodontic treatment is mainly based on root canal disinfection and its failure may be motivated by microbial resistance. Endodontic therapy can be benefitted by host defense peptides (HDPs), which are multifunctional molecules that act against persistent infection and inflammation. This study aimed to evaluate the antimicrobial, cytotoxic and immunomodulatory activity of several HDPs, namely clavanin A, clavanin A modified (MO) and LL-37, compared to intracanal medication Ca(OH)₂. HDPs and Ca(OH)₂ were evaluated by: (1) antimicrobial assays against Candida albicans and Enterococcus faecalis, (2) cytotoxicity assays and (3) cytokine tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-1α, IL-6, IL-10 and IL-12 and nitric oxide (NO) production by RAW 264.7 cells incubated with or without heat-killed (HK) C. albicans or E. faecalis combined or not with interferon-γ. The minimum inhibitory concentration (MIC) was established only for E. faecalis (LL-37, 57 μM). Considering cytotoxicity, clavanin MO was able to reduce cell viability in many groups and demonstrated lowest LC₅₀. The Ca(OH)₂ up-regulated the production of MCP-1, TNF-α, IL-12 and IL-6 and down-regulated IL-1α, IL-10 and NO. Clavanins up-regulated the TNF-α and NO and down-regulated IL-10 production. LL-37 demonstrated up-regulation of IL-6 and TNF-α production and down-regulation in IL-10 and NO production. In conclusion, LL-37 demonstrated better antibacterial potential. In addition, Ca(OH)₂ demonstrated a proinflammatory response, while the HDPs modulated the inflammatory response from non-interference with the active cytokines in the osteoclastogenesis process, probably promoting the health of periradicular tissues.     

抗菌肽的研究进展

抗菌肽是一类抗细菌、真菌、病毒、寄生虫及肿瘤细胞的小分子多肽,是生物抵御自然界中有害微生物侵染的重要因素,且具抗菌谱广、无免疫原性、作用机制独特、耐热性好等特性,有望成为抗生素的替代品。本文着重介绍了抗菌肽的性质、种类、作用机理等方面的研究进展,同时对其应用、目前存在的问题进行了讨论。     

抗菌肽对种植体周围炎主要致病菌生长抑制作用的研究

目的研究抗菌肽KSL及其衍生物KSL—W对种植体周围炎主要致病菌的体外抑菌效果。方法应用二倍稀释法检测KSL和KSL—W对血链球菌、具梭核杆菌和牙龈卟啉单胞菌的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）;MTT法检测KSL和KSL—W对成骨样细胞MG-63的细胞毒性。结果KSL和KSL—W对具梭核杆菌的MIC和MBC分别为0．0156mg／mL和0．0313mg／mL,对牙龈卟啉单胞菌的MIC和MBC分别为0．125mg／mL和0．5mg／mL,在0．5mg／mL的浓度范围内对血链球菌没有抑制作用;KSL和KSL-W在0．5mg／mL的浓度范围内没有细胞毒性。结论KSL和KSL—W没有细胞毒性,对具梭核杆菌和牙龈卟啉单胞菌具有抑制作用。     

Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells

Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world [14]. The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania [7] and [39]. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.     

General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides

Model compounds of modified hydrophobicity (H), hydrophobic moment (μ) and angle subtended by charged residues (Φ) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. H and μ influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters’ influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Φ, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of μ and Φ on both lipid bilayers and cell membranes.     

抗菌肽的作用机制、生物活性及应用研究进展

抗菌肽广泛存在于生物界,是辅助生物机体抵抗外来病原体入侵的重要防御分子。抗菌肽不仅能抑制、杀灭多种细菌,而且具有抗真菌、抗寄生虫、抗病毒、抗肿瘤和免疫调节等生物学活性。抗菌肽的作用机制与传统抗生素不同,不仅具有广谱抗微生物作用,而且不易诱导机体产生耐药性,因此,在治疗临床耐药菌株方面具有极大的开发潜力。     

Overview on the recent study of antimicrobial peptides: Origins, functions, relative mechanisms and application

Antimicrobial peptides (AMPs), which are produced by several species including insects, other animals, micro-organisms and synthesis, are a critical component of the natural defense system. With the growing problem of pathogenic organisms resistant to conventional antibiotics, especially with the emergence of NDM-1, there is increased interest in the pharmacological application of AMPs. They can protect against a broad array of infectious agents, such as bacteria, fungi, parasite, virus and cancer cells. AMPs have a very good future in the application in pharmaceuticals industry and food additive. This review focuses on the AMPs from different origins in these recent years, and discusses their various functions and relative mechanisms of action. It will provide some detailed files for clinical research of pharmaceuticals industry and food additive in application.     

Effect of physicochemical properties of peptides from soy protein on their antimicrobial activity

Antimicrobial peptides (AMPs) kill microbial cells through insertion and damage/permeabilization of the cytoplasmic cell membranes and has applications in food safety and antibiotic replacement. Soy protein is an attractive, abundant natural source for commercial production of AMPs. In this research, explicit solvent molecular dynamics (MD) simulation was employed to investigate the effects of (i) number of total and net charges, (ii) hydrophobicity (iii) hydrophobic moment and (iv) helicity of peptides from soy protein on their ability to bind to lipid bilayer and their transmembrane aggregates to form pores. Interaction of possible AMP segments from soy protein with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPC/POPG) bilayers, a mimic of bacterial cell membrane, was investigated. Pore formation was insensitive to helicity and occurred for hydrophobicity threshold in the range of −0.3–0 kcal/mol, hydrophobic moment threshold of 0.3 kcal/mol, net charge threshold of 2. Though low hydrophobicity and high number of charges help in the formation of water channel for transmembrane aggregates, insertion of peptides with these properties requires overcome of energy barrier, as shown by potential of mean force calculations, thereby resulting in low antimicrobial activity. Experimental evaluation of antimicrobial activity of these peptides against Gram positive L. monocytogenes and Gram negative E. coli as obtained by spot-on-lawn assay was consistent with simulation results. These results should help in the development of guidelines for selection of peptides with antimicrobial activity based on their physicochemical properties.     

Effects of antimicrobial peptides of neutrophils on tumor and normal host cells in culture

We carried out a study of the effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine Bac5 on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic β-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic effects. Bac5 belongs to a group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a nonlytic mechanism. We have shown that while PG1 exerted distinct and fast cytotoxic effects towards most of used tumor cells being in a lesser degree toxic for nontransformed host cells; the proline-rich peptide Bac5 possessed modest cytotoxic activity for all tested cells. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as was previously established for human defensins by Panyutich at al., 1995). Pre-incubation of PG1 with α1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of α1-antitrypsin was reduced after the interaction of the serpin with PG1 (while Bac5 did not affect the antiprotease activity of α1-antitrypsin). We used BODIPY FL-tagged PG1 and Bac5 to study the internalization of the labeled peptides into target cells and their intracellular distribution by confocal microscopy. Bac5-BODIPY (at 5 μ M) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations (1—3 μM) was also able to enter the cells without their damaging. By using flow cytometry we showed that lowering a temperature to 4°C caused a significant decrease in the uptake into K562 and U937 cells for both Bac5-BODIPY and PG1-BODIPY. A decline of target cells metabolism also diminished the process of both peptides internalization but for a lesser degree. In the presence of endocytosis inhibitors the penetration of Bac5-BODIPY and PG1-BODIPY into K562 cells was also reduced, but not completely abolished, suggesting that along with endocytosis process some direct penetration of the peptides across cell membranes takes place. The ability of the peptides to internalize into eukaryotic cells may contribute to the idea of participation of AMPs in varied intracellular events, occurring in normal or malignant host cells, for instance, in the modulation of intracellular serpins activity.     

Analyses of dose-response curves to compare the antimicrobial activity of model cationic alpha-helical peptides highlights the necessity for a minimum of two activity parameters

To assess and compare different model Leu-Lys-containing cationic alpha-helical peptides, their antimicrobial activities were tested against Escherichia coli as target organism over a broad peptide concentration range. The natural cationic alpha-helical peptides magainin 2 and PGLa and the cyclic cationic peptide gramicidin S were also tested between comparison. The dose-response curves differed widely for these peptides, making it difficult to rank them into an activity order over the whole concentration range. We therefore compared five different inhibition parameters from dose-response curves: IC(min) (lowest concentration leading to growth inhibition), IC(50) (concentration that gives 50% growth inhibition), IC(max) (related to minimum inhibition concentration and minimum bactericidal concentration), inhibition concentration factor (IC(F); describing the increase in concentration of the peptide between minimum and maximum inhibition), and activity slope (A(S); related to the Hill coefficient). We found that these parameters were covariant: two of them sufficed to characterize the dose dependence and hence the activity of the peptides. This was corroborated by showing that the dose dependences followed the Hill equation, with a small, constant aberration. We propose that the activity of antimicrobial peptides can readily be characterized by both IC(50) and IC(F) (or A(S)) rather than by a single parameter and discuss how this may relate to investigations into their mechanisms of action.     

Interactions of antimicrobial peptides with Leishmania and trypanosomes and their functional role in host parasitism

Antimicrobial peptides (AMPs) are multifunctional components of the innate systems of both insect and mammalian hosts of the pathogenic trypanosomatids Leishmania and Trypanosoma species. Structurally diverse AMPs from a wide range of organisms have in vitro activity against these parasites acting mainly to disrupt surface-membranes. In some cases AMPs also localize intracellularly to affect calcium levels, mitochondrial function and induce autophagy, necrosis and apoptosis. In this review we discuss the work done in the area of AMP interactions with trypanosomatid protozoa, propose potential targets of AMP activity at the cellular level and discuss how AMPs might influence parasite growth and differentiation in their hosts to determine the outcome of natural infection.     

含抗菌肽酵母工程菌对肉仔鸡肠道菌群和免疫功能的影响

目的 研究含抗菌肽（Cec Md、3cs、3js）酵母工程菌对肉仔鸡肠道菌群和免疫功能的影响。方法 采用随机试验设计的方法,选用1日龄健康爱拔益加肉仔鸡400只,随机分成5组,每组设4个重复,每个重复20只鸡（雌雄各半）。空白对照组常规饮水,抗生素组在饮水中添加50 mg/L的泰乐菌素,抗菌肽实验组分别在饮水中添加8 mg/L含Cec Md、3cs和3js酵母工程菌,实验期42 d。结果 （1）Cec Md组的第1、4和5周及3cs组第3、5周的大肠埃希菌数量显著低于空白对照组（P<0.05）;Cec Md组和3cs组的第2至第6周及3js组的第2至第4周沙门菌数量显著低于空白对照组（P<0.05）;Cec Md组的第4至第6周及3cs组和3js组的第5周的乳杆菌数量显著高于空白对照组（P<0.05）;Cec Md组的第2、3、5周和6周及3cs组第5、6周及3js组的第3至第6周的双歧杆菌数量显著高于空白对照组（P<0.05）。（2）Cec Md组的第3、5周及3cs和3js组的第6周的IgA含量显著高于空白对照组（P<0.05）;Cec Md组和3js组的第3周的IgG含量显著高于空白对照组（P<0.05）。3js组的第1周的IgM含量显著高于空白对照组（P<0.05）,Cec Md组第4、5周的IgE含量显著高于空白对照组（P<0.05）。结论 含抗菌肽Cec Md、3cs和3js酵母工程菌可以抑制肉仔鸡肠道有害菌群的生长,促进其有益菌群的增殖,且可以改善肉仔鸡的免疫功能。