Mycoplasma pneumoniae infection induces a neutrophil-derived antimicrobial peptide, cathelin-related antimicrobial peptide

In innate immunity, cationic antimicrobial peptides including cathelin-related antimicrobial peptide (CRAMP) are known to play critical roles in protecting the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between CRAMP and mycoplasmas. In the present study, the antimicrobial activity of CRAMP against M. pneumoniae and the expression of CRAMP in bronchoalveolar lavage fluid (BALF) of M. pneumoniae-infected mice was examined. CRAMP at 10-20 μg/mL reduced the growth of two strains of M. pneumoniae by 100 to 1000-fold. The amount of CRAMP in the BALF of M. pneumoniae-infected mice was 20～25 ng/mL by ELISA. The presence of mature CRAMP in BALF was observed by Western blotting. Neutrophils in BALF showed a fair amount of CRAMP in their cytoplasm by immunofluorescence. Furthermore, the addition of M. pneumoniae resulted in the release of a large amount of CRAMP from neutrophils induced by thioglycolate. These results suggest that CRAMP from neutrophils may play an important role in protection against M. pneumoniae infection.     

Lactoferricin, a new antimicrobial peptide

Lactoferricin B (LF-B) is a peptide derived from acid-pepsin digestion of bovine lactoferrin, which has antimicrobial properties. In order to assess the antimicrobial spectrum of LF-B and its possible in vivo uses, the minimum inhibitory and microbicidal concentrations of pure lactoferricin B were determined for a range of bacterial species and under varying conditions of growth including growth phase and size of the inoculum, pH and ionic strength of the medium. Lactoferricin B was bactericidal against a wide range of bacteria and Candida albicans. Proteus spp., Pseudomonas cepacia and Serratia spp. were resistant. The bactericidal activity of LF-B was inhibited by increasing ionic strength and bacterial inoculum and at acid pH. The activity of lactoferricin B was completely inhibited by the addition of 5% whole cow's milk and was reduced in the presence of increasing concentrations of mucin. These results indicate the potential of LF-B to reduce the numbers of organisms in a simple medium, but raise doubts about its role in vivo because of its sensitivity to changes in physical variables. It may be that lactoferricin exerts a transient antimicrobial effect at mucosal surfaces.     

Purification and characterization of the antimicrobial peptide, ostricacin

An antimicrobial peptide, ostricacin-1, has been purified and characterized from ostrich leukocytes. The peptide has a mass of 4011 and contained 36 residues, including 3 intramolecular cystine disulfide bonds. Ostricacin-1 has a primary sequence homology to the -defensin family and was active at 6.7 g ml^–1 against E. coli and Staphylocccus aureus in vitro.     

抗菌肽药物工程化技术研究进展

尽管抗生素在畜牧业疾病防治中的主导地位在短期内不会动摇,但病原菌耐药性形成与快速发展让抗菌肽成为近年新药研发热点之一。作为一种在生物体内广泛存在的天然免疫物质,抗菌肽具有抗菌、抗病毒、抗肿瘤和免疫调节等功能,由于药效短、对蛋白酶敏感、细胞毒性高等缺陷而限制其应用。本文综述了抗菌肽基因工程技术、聚乙二醇(PEG)化、靶向性改造和固定化等工程技术在抗菌肽新药开发中的应用研究进展,以促进抗菌肽产业化。     

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.     

Effect of amidation on the antimicrobial peptide aurein 2.5 from Australian southern bell frogs

Aurein 2.5 is a naturally C-terminally amidated amphibian antimicrobial peptide. C-terminal amidation can increase efficacy and hence a comparison was made between aurein 2.5-CONH2 and its nonamidated analogue. Amidation of the C-terminal carboxyl of aurein 2.5 enhanced antimicrobial activity 2.5- fold against Klebsiella pneumonia. Our results demonstrate that both peptide analogues had high surface activities (23 mN m-1for aurein 2.5-COOH and 26 mN m-1 aurein 2.5-CONH2). Circular dichroism measurements suggest that the helical content of the amidated form, in the presence of trifluoroethanol, was significantly enhanced (33.66 % for aurein 2.5-COOH and 60.89 % aurein 2.5-CONH2). The interaction of aurein 2.5 with bacterial cell membrane mimics was investigated using Langmuir monolayers. Aurein 2.5-CONH2 induced stable surface pressure changes in monolayers formed from K. pneumonia (circa 4.7 mN m-1), however, lower surface pressure changes were observed for aurein 2.5- COOH (circa 3.8 mN m-1). The data shows that in the case of aurein 2.5, amidation is able to enhance antibacterial activity and it is proposed that the increase in effectiveness is due to stabilization of the α-helical structure at the membrane interface.     

Effect of a phorbol ester on basic surface properties of trichomonads

The effect of nanomolar concentrations of 12-O-tetradecanoil-phorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoaTrichomonas vaginalis andTritrichomonas foetus was evaluated by means of measurements of the parasites’ surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites’ net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.     

Effect of a phorbol ester on basic surface properties of trichomonads.

The effect of nanomolar concentrations of 12-O-tetradecanoilphorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoa Trichomonas vaginalis and Tritrichomonas foetus was evaluated by means of measurements of the parasites' surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites' net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.     

Effect of the antimicrobial peptide indolicidin on the green peach aphid Myzus persicae (Sulzer)

Abstract:  The green-peach aphid, Myzus persicae (Sulzer) (Hem., Aphididae), is a major agricultural pest of a wide range of host plants, causing damage by feeding and indirectly by transmitting viruses. In this study we tested the effect of the antimicrobial peptide indolicidin on M. persicae survival and on its essential bacterial endosymbionts. Artificial diet bioassays showed a significant dose-dependent lethal response of indolicidin on M. persicae survival (LD₅₀ of 209 ± 60  μ g/ml). Histological analysis of indolicidin-treated aphids revealed a lower number of distorted mycetocytes, whereas control aphids showed abundant number of rounded and filled mycetocytes. These results suggest that aphid survival could be affected via reduction of its endosymbionts. Thus, aphid control based on antimicrobial substances against endosymbionts could be a promising strategy that needs to be further explored.     

Structure-function relationships in the tryptophan-rich, antimicrobial peptide indolicidin.

Indolicidin is a cationic 13 amino acid peptide amide produced in the granules of bovine neutrophils with the sequence H-ILPWKWPWWPWRR-NH2. Indolicidin is both antimicrobial and, to a lesser extent, haemolytic. In order to systematically investigate structure-function relationships, the solid-phase synthesis of indolicidin and 48 distinct analogues are reported, as well as the characterization of their respective biological properties. Peptides synthesized and characterized include analogues with modified terminal functions, truncations from either terminus, an alanine scan to determine the role of each individual amino acid, specific amino acid exchanges of aromatic, charged and structural residues and several retro-, inverso- and retroinverso-analogues. Together, characterization of these analogues identifies specific residues involved in antimicrobial or haemolytic activity and suggests a core structure that may form a scaffold for the further development of peptidomimetic analogues of indolicidin.     

Antifungal activity of synthetic peptide derived from halocidin, antimicrobial peptide from the tunicate, Halocynthia aurantium

Halocidin is an antimicrobial peptide isolated from the hemocytes of the tunicate. Among the several known synthetic halocidin analogues, di-K19Hc has been previously confirmed to have the most profound antibacterial activity against antibiotic-resistant bacteria. This peptide has been considered to be an effective candidate for the development of a new type of antibiotic. In this study, we have assessed the antifungal activity of di-K19Hc, against a panel of fungi including several strains of Aspergillus and Candida. As a result, we determined that the MICs of di-K19Hc against six Candida albicans and two Aspergillus species were below 4 and 16 microg/ml, respectively, thereby indicating that di-K19Hc may be appropriate for the treatment of several fungal diseases. We also conducted an investigation into di-K19Hc's mode of action against Candida albicans. Our colony count assay showed that di-K19Hc killed C. albicans within 30s. Di-K19Hc bound to the surface of C. albicans via a specific interaction with beta-1,3-glucan, which is one of fungal cell wall components. Di-K19Hc also induced the formation of ion channels within the membrane of C. albicans, and eventually observed cell death, which was confirmed via measurements of the K+ released from C. albicans cells which had been treated with di-K19Hc, as well as by monitoring of the uptake of propidium iodide into the C. albicans cells. This membrane-attacking quality of di-K19Hc was also visualized via confocal laser and scanning electron microscopy.     

Hepcidin, a urinary antimicrobial peptide synthesized in the liver

Cysteine-rich antimicrobial peptides are abundant in animal and plant tissues involved in host defense. In insects, most are synthesized in the fat body, an organ analogous to the liver of vertebrates. From human urine, we characterized a cysteine-rich peptide with three forms differing by amino-terminal truncation, and we named it hepcidin (Hepc) because of its origin in the liver and its antimicrobial properties. Two predominant forms, Hepc20 and Hepc25, contained 20 and 25 amino acid residues with all 8 cysteines connected by intramolecular disulfide bonds. Reverse translation and search of the data bases found homologous liver cDNAs in species from fish to human and a corresponding human genomic sequence on human chromosome 19. The full cDNA by 5' rapid amplification of cDNA ends was 0.4 kilobase pair, in agreement with hepcidin mRNA size on Northern blots. The liver was the predominant site of mRNA expression. The encoded prepropeptide contains 84 amino acids, but only the 20-25-amino acid processed forms were found in urine. Hepcidins exhibited antifungal activity against Candida albicans, Aspergillus fumigatus, and Aspergillus niger and antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and group B Streptococcus. Hepcidin may be a vertebrate counterpart of cysteine-rich antimicrobial peptides produced in the fat body of insects.     

Effect of the antimicrobial peptide gomesin against different life stages of Plasmodium spp

While seeking strategies for interfering with Plasmodium development in vertebrate/invertebrate hosts, we tested the activity of gomesin, an antimicrobial peptide isolated from the hemocytes of the spider Acanthoscurria gomesiana. Gomesin was tested against asexual, sexual and pre-sporogonic forms of Plasmodium falciparum and Plasmodium berghei parasites. The peptide inhibited the in vitro growth of intraerythrocytic forms of P. falciparum.When gomesin was added to in vitro culture of P. berghei mature gametocytes, it significantly inhibited the exflagellation of male gametes and the formation of ookinetes. In vivo, the peptide reduced the number of oocysts of both Plasmodium species in Anopheles stephensi mosquitoes, and did not appear to affect the mosquitoes. These properties make gomesin an excellent candidate as a transmission blocking agent for the genetic engineering of mosquitoes.     

Salt-resistant homodimeric bactenecin, a cathelicidin-derived antimicrobial peptide

The cathelicidin antimicrobial peptide bactenecin is a beta-hairpin molecule with a single disulfide bond and broad antimicrobial activity. The proform of bactenecin exists as a dimer, however, and it has been proposed that bactenecin is released as a dimer in vivo, although there has been little study of the dimeric form of bactenecin. To investigate the effect of bactenecin dimerization on its biological activity, we characterized the dimer's effect on phospholipid membranes, the kinetics of its bactericidal activity, and its salt sensitivity. We initially synthesized two bactenecin dimers (antiparallel and parallel) and two monomers (beta-hairpin and linear). Under oxidative folding conditions, reduced linear bactenecin preferentially folded into a dimer forming a ladder-like structure via intermolecular disulfide bonding. As compared to the monomer, the dimer had a greater ability to induce lysis of lipid bilayers and was more rapidly bactericidal. Interestingly, the dimer retained antimicrobial activity at physiological salt concentrations (150 mm NaCl), although the monomer was inactivated. This salt resistance was also seen with bactenecin dimer containing one intermolecular disulfide bond, and the bactenecin dimer appears to undergo multimeric oligomerization at high salt concentrations. Overall, dimeric bactenecin shows potent and rapid antimicrobial activity, and resists salt-induced inactivation under physiological conditions through condensation and oligomerization. These characteristics shed light on the features that a peptide would need to serve as an effective therapeutic agent.     

Structure of the antimicrobial peptide tachystatin A

The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded beta-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist omega-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that omega-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and omega-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and omega-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and omega-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe(9) of tachystatin A might be an essential residue for binding to chitin.     

Effect of head group and curvature on binding of the antimicrobial peptide tritrpticin to lipid membranes

In this work we examine the interaction between the 13-residue cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) and model membranes of variable lipid composition. The effect on peptide conformational properties was investigated by means of CD (circular dichroism) and fluorescence spectroscopies. Based on the hypothesis that the antibiotic acts through a mechanism involving toroidal pore formation, and taking into account that models of toroidal pores imply the formation of positive curvature, we used large unilamellar vesicles (LUV) to mimic the initial step of peptide-lipid interaction, when the peptide binds to the bilayer membrane, and micelles to mimic the topology of the pore itself, since these aggregates display positive curvature. In order to more faithfully assess the role of curvature, micelles were prepared with lysophospholipids containing (qualitatively and quantitatively) head groups identical to those of bilayer phospholipids. CD and fluorescence spectra showed that, while TRP3 binds to bilayers only when they carry negatively charged phospholipids, binding to micelles occurs irrespective of surface charge, indicating that electrostatic interactions play a less predominant role in the latter case. Moreover, the conformations acquired by the peptide were independent of lipid composition in both bilayers and micelles. However, the conformations were different in bilayers and in micelles, suggesting that curvature has an influence on the secondary structure acquired by the peptide. Fluorescence data pointed to an interfacial location of TRP3 in both types of aggregates. Nevertheless, experiments with a water soluble fluorescence quencher suggested that the tryptophan residues are more accessible to the quencher in micelles than in bilayers. Thus, we propose that bilayers and micelles can be used as models for the two steps of toroidal pore formation.     

细菌对抗菌肽的耐受机制

抗菌肽广谱、高特异、高生物活性等特点决定其具极大的临床应用潜力,然而抗菌肽的耐受是其药物开发必须重视和亟待克服的问题。从生物学的观点看,部分细菌可以产生抗菌肽,其必定存在逃避自身抗菌肽作用的机制;从进化的观点看,宿主和病原体之间是相互抑制、相互逃避、相互适应的关系,细菌在漫长的进化中会形成应对抗菌肽的特殊机制。抗菌肽对细菌存在多种作用机制,其核心是依赖于与细胞膜相互作用或进入细胞,进而改变膜完整性或干扰胞内生理生化反应导致细菌死亡;而细菌通过减弱抗菌肽结合、降低抗菌肽有效浓度等方式产生对抗菌肽的耐受。这些耐受机制也为抗菌肽类药物开发提供重要的启示。     

Effect of phosphatidylcholine bilayer thickness and molecular order on the binding of the antimicrobial peptide maculatin 1.1

Antimicrobial peptides (AMPs) interact directly with bacterial membrane lipids. Thus, changes in the lipid composition of bacterial membranes can have profound effects on the activity of AMPs. In order to understand the effect of bilayer thickness and molecular order on the activity of AMPs, the interaction of maculatin 1.1 (Mac1.1) with phosphatidylcholine (PC) model membranes composed of different monounsaturated acyl chain lengths between 14 and 22 carbons was characterised by dual polarisation interferometry (DPI) and 31P and 1H solid-state NMR techniques. The thickness and bilayer order of each PC bilayer showed a linear dependence on the acyl chain length. The binding of Mac1.1 exhibited a biphasic dependency between the amount of bound Mac1.1 and bilayer thickness, whereby the mass of bound peptide increased from C14 to C16 and then decreased from C16 to C22. Significant perturbation of 31P chemical shift anisotropy (CSA) values was only observed for DOPC (C18) and DEPC (C22), respectively. In the case of DEPC, the greater range in CSA indicated different headgroup conformations or environments in the presence of Mac1.1. Overall, the results indicated that there is a significant change in the bilayer order upon binding of Mac1.1 and this change occurred in a co-operative manner at higher concentrations of Mac1.1 with increasing bilayer thickness and order. Overall, an optimum bilayer thickness and lipid order may be required for effective membrane perturbation by Mac1.1 and increasing the bilayer thickness and order may counteract the activity of Mac1.1 and play a role in antimicrobial resistance to AMPs.     

抗菌肽耐药性研究进展

抗菌肽是生物体产生的一类具有抵抗外源性病原体功能的小分子多肽,具有抗细菌、真菌、病毒、癌细胞等多种活性.近几年的研究发现细菌会对抗菌肽产生耐药性.本文就细菌的构成性耐药性机制和诱导性耐药性机制等研究进展进行综述.     

Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33

With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides (AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5?mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1?l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195?Da). The recombinant peptide LFT33 caused an increase in antimicrobial activity (IC(50)?=?16-64?μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.