Identification and characterization of two novel antimicrobial peptides,temporin‐Ra and temporin‐Rb,from skin secretions of the marsh frog (Rana ridibunda)

In this study, two novel antimicrobial peptides from the skin secretions of the marsh frog, Rana ridibunda, named temporin‐Ra and temporin‐Rb, were identified and purified using RP‐HPLC. Temporin‐Ra and temporin‐Rb are composed of 14 and 12 amino acids, respectively. Our results show that these peptides have inhibitory effects on both gram‐negative and gram‐positive bacteria, especially antibiotic resistant strains prevalent in hospitals, such as Staphylococcus aureus and Streptococcus agalactiae. The sequences and molecular weights of these peptides were determined using tandem MS. The molecular masses were found to be 1242.5 Da for temporin‐Rb and 1585.1 Da for temporin‐Ra. Human red blood cells tolerated well exposure to temporin‐Ra and temporin‐Rb, which, at a concentration of 60 µg/ml, induced 1.3% and 1.1% hemolysis, respectively. MIC values of these peptides are suitable for potent antimicrobial peptides. The low hemolytic effect and wide‐spectrum antimicrobial activity suggest a possible therapeutic application of these novel peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A Novel Antimicrobial Peptide Derived from the Insect Paederus dermatitis

Much research has been focused on antimicrobial peptides (AMPs) derived from insect immune defense reactions due to their potential in the development of new antibiotics. In this study, a new AMP from the insect Paederus dermatitis, named sarcotoxin Pd was identified and purified using gel filtration and reverse-phase high-performance liquid chromatography. Our results showed that this peptide has broad-spectrum inhibitory effects on examined microbes. Sarcotoxin Pd is composed of 34 amino acids and its molecular weight was estimated to be 3613.26 ± 0.5 Da. Minimum inhibitory concentration (MIC) values of sarcotoxin Pd against Gram-negative bacteria were lower than Gram-positive bacteria and fungi. The identified peptide showed the highest antimicrobial effect against Klebsiella pneumonia and Escherichia coli. This peptide did not reveal significant hemolytic activity against human red blood cells particularly in the range of MIC values. Confirming the potential antimicrobial activities of synthetic peptide, this paper addresses the role of sarcotoxin Pd in the treatment of systemic microbial illnesses.     

A novel cysteine-rich antimicrobial peptide from the mucus of the snail of Achatina fulica

Antimicrobial peptides (AMPs) are important components of the innate immunity. Many antimicrobial peptides have been found from marine mollusks. Little information about AMPs of mollusks living on land is available. A novel cysteine-rich antimicrobial peptide (mytimacin-AF) belonging to the peptide family of mytimacins was purified and characterized from the mucus of the snail of Achatina fulica. Its cDNA was also cloned from the cDNA library. Mytimacin-AF is composed of 80 amino acid residues including 10 cysteines. Mytimacin-AF showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria and the fungus Candida albicans. Among tested microorganisms, it exerted strongest antimicrobial activity against Staphylococcus aureus with a minimal peptide concentration (MIC) of 1.9 μg/ml. Mytimacin-AF had little hemolytic activity against human blood red cells. The current work confirmed the presence of mytimacin-like antimicrobial peptide in land-living mollusks.     

Production,purification, and characterization of the cecropin from <Emphasis Type="Italic">Plutella xylostella</Emphasis>, pxCECA1, using an intein-induced self-cleavable system in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD–intein–pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.     

Membrane-active antimicrobial peptide identified in Rana arvalis by targeted DNA sequencing

Antimicrobial peptides (AMPs) are naturally produced, gene encoded molecules with a direct antimicrobial activity against pathogens, often also showing other immune-related properties. Anuran skin secretions are rich in bioactive peptides, including AMPs, and we have reported a novel targeted sequencing approach to identify novel AMPs simultaneously in different frog species, from small quantities of skin tissue. Over a hundred full-length peptides were identified from specimens belonging to five different Ranidae frog species, out of which 29 were novel sequences. Six of these were selected for synthesis and testing against a panel of Gram-negative and Gram-positive bacteria. One peptide, identified in Rana arvalis, proved to be a potent and broad-spectrum antimicrobial, active against ATCC bacterial strains and a multi-drug resistant clinical isolate. CD spectroscopy suggests it has a helical conformation, while surface plasmon resonance (SPR) that it may self-aggregate/oligomerize at the membrane surface. It was found to disrupt the bacterial membrane at sub-MIC, MIC and above-MIC concentrations, as observed by flow cytometry and/or visualized by atomic force microscopy (AFM). Only a limited toxicity was observed towards peripheral blood mononuclear cells (PBMC) with a more pronounced effect observed against the MEC-1 cell line.     

Ruminococcin C, a new anti-Clostridium perfringens bacteriocin produced in the gut by the commensal bacterium Ruminococcus gnavus E1

When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized.     

Purification and cDNA Cloning of Antimicrobial Peptides from the Skin Secretion of the Chinese Frog Rana chensinensis

The skin secretions of amphibians are a rich source of bioactive peptides. We isolated chensirin-1 and chensirin-2 from the skin secretion of the Chinese frog Rana chensinensis. Sephadex-G-50 and RP-HPLC were employed to purify these peptides. The amino acid sequences of these peptides were VLPLVGNLLNDLLGE and IIPLPLGYFAKKT, respectively, as determined by Edman degradation. The molecular weights were 1578.7 and 1460.8 Da, respectively, as analyzed by HPLC-ESI-MS. The chensirin cDNA was cloned by 5′ and 3′ amplification of cDNA ends, synthesized and purified. The antibacterial activities of the chensirins were tested using minimum inhibitory concentration, the results indicated that chensirins inhibit the growth of gram-negative and gram-positive bacteria. Among them, chensirin-1 is a novel peptide with a higher antibacterial activity compared to other similar antimicrobial peptides. These low molecular weight peptides with good antimicrobial efficacy are considered potential sources for developing new antimicrobial agents to improve traditional drug resistance.

     

Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Pseudomonas aeruginosa is one of the major nosocomial pathogen that can causes a wide variety of acute and chronic infections P. aeruginosa is a dreaded bacteria not just because of the high intrinsic and acquired antibiotic resistance rates but also the biofilm formation and production of multiple virulence factors. We investigated the in vitro activities of antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin and colistin) and antimicrobial cationic peptides (AMPs; LL-37, CAMA: cecropin(1–7)-melittin A(2–9) amide, melittin, defensin and magainin-II) alone or in combination against biofilms of laboratory strain ATCC 27853 and 4 clinical strains of P. aeruginosa. The minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentrations (MBEC) were determined by microbroth dilution technique. The MBEC values of antibiotics and AMPs were 80–>5120 and 640–>640 mg/L, respectively. When combined with the LL-37 or CAMA at 1/10× MBEC, the MBEC values of antibiotics that active against biofilms, were decreased up to 8-fold. All of the antibiotics, and AMPs were able to inhibit the attachment of bacteria at the 1/10× MIC and biofilm formation at 1× or 1/10× MIC concentrations. Time killing curve studies showed 3-log₁₀ killing against biofilms in 24 h with almost all studied antibiotics and AMPs. Synergism were seen in most of the studied combinations especially CAMA/LL-37 + ciprofloxacin against at least one or two strains’ biofilms. Since biofilms are not affected the antibiotics at therapeutic concentrations, using a combination of antimicrobial agents including AMPs, or inhibition of biofilm formation by blocking the attachment of bacteria to surfaces might be alternative methods to fight with biofilm associated infections.     

Purification and identification of antioxidant peptides from the skin protein hydrolysate of two marine fishes,horse mackerel (<Emphasis Type="Italic">Magalaspis cordyla</Emphasis>) and croaker (<Emphasis Type="Italic">Otolithes ruber</Emphasis>)

In the current study, two peptides with antioxidant properties were purified from skin protein hydrolysates of horse mackerel (Magalaspis cordyla) and croaker (Otolithes ruber) by consecutive chromatographic fractionations including ion exchange chromatography and gel filtration chromatography. By electron spray ionization double mass spectrometry (ESI-MS/MS), the sequence of the peptide from the skin protein hydrolysate of horse mackerel was identified to be Asn-His-Arg-Tyr-Asp-Arg (856 Da) and that of croaker to be Gly-Asn-Arg-Gly-Phe-Ala-Cys-Arg-His-Ala (1101.5 Da). The antioxidant activity of these peptides was tested by electron spin resonance (ESR) spectrometry using 1-diphenyl-2-picryl hydrazyl (DPPH^·) and hydroxyl (OH^·) radical scavenging assays. Both peptides exhibited higher activity against polyunsaturated fatty acid (PUFA) peroxidation than the natural antioxidant α-tocopherol. These results suggest that the two peptides isolated from the skin protein hydrolysates of horse mackerel and croaker are potent antioxidants and may be effectively used as food additives and as pharmaceutical agents.     

Isolation and Chemical Characterization of an Alpha-Helical Peptide,Dendrocin-ZM1, Derived from Zataria multiflora Boiss with Potent Antibacterial Activity

Today, resistance of microorganisms to antibiotics has become a major challenge. To overcome this problem, development of new drugs, besides research on their antibacterial activity, is essential. Among chemical components, antimicrobial peptides (AMPs) exhibit antibacterial activity and can be selected as suitable antimicrobial candidates. In this study, a novel antimicrobial peptide, called dendrocin-ZM1, with a molecular weight of?~3716.48 Da, was isolated from Zataria multiflora Boiss (ZM) and purified via precipitation with ammonium sulfate and reverse-phase HPLC chromatography; it was then sequenced via Edman degradation. The in silico method was used to examine the physicochemical properties of dendrocin-ZM1. In this study, four reference strains (gram-positive and gram-negative) and one clinical vancomycin-resistant Staphylococcus aureus strain were used to survey the antimicrobial activities. Moreover, to examine cytotoxicity and hemolytic activity, a HEK-293 cell line and human red blood cells (RBCs) were used, respectively. Evaluation of the physicochemical properties of dendrocin-ZM1, as an AMP, indicated a net charge of?+?7 and a hydrophobicity percentage of 54%. This peptide had an amphipathic alpha-helical conformation. It exhibited broad-spectrum antibacterial activities against the tested strains at minimum inhibitory concentrations (MICs) of 4–16 μg/mL. Besides, this peptide showed negligible hemolysis and cytotoxicity in the MIC range. It also exhibited heat stability at temperatures of 20 to 80 °C and was active in a broad pH range (from 6.0 to 10.0). Overall, the present results suggested dendrocin-ZM1 as a remarkable antimicrobial candidate.

     

Antimicrobial peptides derived from different animals: comparative studies of antimicrobial properties,cytotoxicity and mechanism of action

Animals posses a large variety of antimicrobial peptides (AMPs) that serve as effective components in innate host defenses against microbial infections. These antimicrobial peptides differ in amino acid composition, range of antimicrobial specificities, hemolysis, cytotoxicity and mechanisms of action. This study was designed to evaluate their therapeutic potential of the following six antimicrobial peptides initially found from animals: cecropin P1, indolicidin, LL-37, palustrin-OG1, LFP-20 and LFB-11. Our results indicated that cecropin P1 possessed the most desired biological activity, with fast and potent antimicrobial activity but only slight hemolytic or cytotoxic activity against human cells. Indolicidin was more effective against gram-positive bacteria but with higher hemolytic and cytotoxic activity on human peripheral blood mononuclear cell (PBMCs) (P < 0.05). Although LFP-20 and LFB-11 had moderate activity against tested strains and need 30 min to kill E. coli, they showed almost no hemolytic and cytotoxic activity towards PBMCs (P < 0.01). Indolicidin could form pores of well-defined structure in bacterial membranes whereas lysis of E. coli cells was observed after addition LFB-11 and LL-37 at 1 × MIC for 1 h. LL-37 treatment could lead to the leakage of entire bacterial cytoplasmic contents. The most obvious phenomenon was protuberant structures on the E. coli cell surface after incubation with LFP-20, cecropin P1 and palustrin-OG1. The results presented here illustrate that AMPs derived from different animals exhibited different antimicrobial characteristics. Because of their potent and broad-spectrum antimicrobial activity, low cytotoxicity towards normal cells, and the unique mechanism of action, these peptides may provide the impetus for the development of novel strategies for the prevention of bacterial infections in animals.     

Antimicrobial activity of four cationic peptides immobilised to poly-hydroxyethylmethacrylate

The objective of this study was to immobilise and characterise a variety of antimicrobial peptides (AMPs) onto poly-hydroxyethylmethacrylate (pHEMA) surfaces to achieve an antibacterial effect. Four AMPs, viz. LL-37, melimine, lactoferricin and Mel-4 were immobilised on pHEMA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which assisted covalent attachment. Increasing concentrations of AMPs were immobilised to determine the effect on the adhesion of Pseudomonas aeruginosa and Staphylococcus aureus. The AMP immobilised pHEMAs were characterised by X-ray photoelectron spectroscopy (XPS) to determine the surface elemental composition and by amino acid analysis to determine the total amount of AMP attached. In vitro cytotoxicity of the immobilised pHEMA samples to mouse L929 cells was investigated. Melimine and Mel-4 when immobilised at the highest concentrations showed 3.1 ± 0.6 log and 1.3 ± 0.2 log inhibition against P. aeruginosa, and 3.9 ± 0.6 log and 2.4 ± 0.5 log inhibition against S. aureus, respectively. Immobilisation of LL-37 resulted in up to 2.6 ± 1.0 log inhibition against only P. aeruginosa, but no activity against S. aureus. LFc attachment showed no antibacterial activity. Upon XPS analysis, immobilised melimine, LL-37, LFc and Mel-4 had 1.57 ± 0.38%, 1.13 ± 1.36%, 0.66 ± 0.47% and 0.73 ± 0.32% amide nitrogen attached to pHEMA compared to 0.12 ± 0.14% in the untreated controls. Amino acid analysis determined that the total amount of AMP attachment to pHEMA was 44.3 ± 7.4 nmol, 3.8?±?0.2?nmol, 6.5 ± 0.6 nmol and 48.9 ± 2.3 nmol for the same peptides respectively. None of the AMP immobilised pHEMA surfaces showed any toxicity towards mouse L929 cells. The immobilisation of certain AMPs at nanomolar concentration to pHEMA is an effective option to develop a stable antimicrobial surface.     

Piscidin 4, a novel member of the piscidin family of antimicrobial peptides

Piscidins are linear, amphipathic, antimicrobial peptides (AMPs) with broad, potent, activity spectrum. Piscidins and other members of the piscidin family appear to comprise the most common group of AMPs in teleost fish. All piscidins and related members of the piscidin family described to date are 18–26 amino acids long. We report here the isolation of a novel 5329.25 Da, 44-residue (FFRHLFRGAKAIFRGARQGXRAHKVVSRYRNRDVPETDNNQEEP) antimicrobial peptide from hybrid striped bass (Morone chrysops female x M. saxatilis male). We have named this peptide “piscidin 4” since it has considerable (to > 65%) N-terminal sequence homology to piscidins 1–3 and this distinctive, 10 to 11-residue, N-terminus is characteristic of piscidins. The native peptide has a modified amino acid at position 20 that, based upon mass spectrometry data, is probably a hydroxylated tryptophan. Synthetic piscidin 4 (with an unmodified tryptophan at position 20) has similar antibacterial activity to that of the native peptide. Piscidin 4 demonstrates potent, broad-spectrum, antibacterial activity against a number of fish and human pathogens, including multi-drug resistant bacteria. Its potent antimicrobial activity suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass.     

Novel families of antimicrobial peptides with multiple functions from skin of Xizang plateau frog, Nanorana parkeri

Xizang plateau frog (Nanorana parkeri) captured in Lhasa, Tibet, China, solely lives in the subtropical plateau, where there is strong ultraviolet radiation and long duration of sunshine. Considering its harsh living environment, the frog's innate defense against microbes and environmental stress was investigated. In current study, three antimicrobial peptides (AMPs) were purified and characterized from the skin secretion of N. parkeri. The coding cDNA sequences were also cloned from the skin cDNA library of N. parkeri. By structural characterization, two peptides were identified belonging to Japonicin-1 family, and named as Japonicin-1Npa (FLLFPLMCKIQGKC) and Japonicin-1Npb (FVLPLVMCKILRKC). The third peptide isolated named Parkerin with a unique sequence of GWANTLKNVAGGLCKITGAA did not show similarity to any known amphibian AMPs. Multi-functions of three AMPs were examined (antioxidant, MCD, hemolytic etc). Their solution structures determined by CD and antimicrobial mechanisms investigated by SEM are very well consistent with their functional characters. Current result suggests that these novel multi-functional AMPs could play an important role in defending N. parkeri against environmental oxidative stress and pathogenic microorganisms, which may partially reveal the ecological adaptation of these plateau-living amphibians.     

An immunological method for quantifying antibacterial activity in Salmo salar (Linnaeus, 1758) skin mucus

Antimicrobial peptides (AMPs) are a pivotal component of innate immunity in lower vertebrates. The aim of this study was to develop an immunological method for quantifying AMPs in Salmo salar skin mucus. A known antimicrobial peptide derived from histone H1 previously purified and described from S. salar skin mucus (SAMP H1) was chemically synthesized and used to obtain antibodies for the quantification of the molecule via ELISA. Using skin mucus samples, a correlation of bacterial growth inhibition versus SAMP H1 concentration (ELISA) was established. The results provide the first evidence for quantifying the presence of active AMPs in the skin mucus of S. salar through the use of an immunological method.     

Peptidomics and genomics analysis of novel antimicrobial peptides from the frog,Rana nigrovittata

Much attention has been paid on amphibian peptides for their wide-ranging pharmacological properties, clinical potential, and gene-encoded origin. More than 300 antimicrobial peptides (AMPs) from amphibians have been studied. Peptidomics and genomics analysis combined with functional test including microorganism killing, histamine-releasing, and mast cell degranulation was used to investigate antimicrobial peptide diversity. Thirty-four novel AMPs from skin secretions of Rana nigrovittata were identified in current work, and they belong to 9 families, including 6 novel families. Other three families are classified into rugosin, gaegurin, and temporin family of amphibian AMP, respectively. These AMPs share highly conserved preproregions including signal peptides and spacer acidic peptides, while greatly diversified on mature peptides structures. In this work, peptidomics combined with genomics analysis was confirmed to be an effective way to identify amphibian AMPs, especially novel families. Some AMPs reported here will provide leading molecules for designing novel antimicrobial agents.     

Design and Synthesis of Lipopolysaccharide-Binding Antimicrobial Peptides Based on Truncated Rabbit and Human CAP18 Peptides and Evaluation of Their Action Mechanism

Lipopolysaccharide (LPS) is a toxic and immunogenic agent for human. Additionally, LPS is a good target for some antimicrobial compounds, including antimicrobial peptides (AMPs). LPS-binding peptides (LBPs) can recognize and neutralize LPS. Rabbit and human cathelicidins are AMPs with LPS-binding activity. In this study, we designed and synthesized two new truncated LBPs from rabbit and human CAP18 peptides by in silico methods. After synthesis of peptides, the antimicrobial properties and LPS-binding activity of these peptides were evaluated. The parental rabbit and human CAP18 peptides were selected as positive controls. Next, the changes in the secondary structure of these peptides before and after treatment with LPS were measured by circular dichroism (CD). Human cytotoxicity of the peptides was evaluated by MTT and red blood cells (RBCs) hemolysis assays. Finally, field emission scanning electron microscopy (FE-SEM), confocal microscopy, and flow cytometry were performed to study the action mechanism of these peptides. Results indicated that the hCap18 and rCap18 had antibacterial activity (at a MIC of 4–128 μg/mL). The results of the quantitative LAL test demonstrated that LPS-binding activity of hCap18 peptide was better than rCap18, while rCap18 peptide had better antimicrobial properties. Furthermore, rCap18 had less cytotoxicity than hCap18. However, both peptides were nontoxic for normal human skin fibroblast cell in MIC range. In conclusion, rCap18 has good antibacterial properties, while hCap18 can be tested as a diagnostic molecule in our future studies.

     

Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 urethritis clade: mutations in the pilMNOPQ operon

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non‐encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64–256 µg ml^–1). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384–1024 µg ml^–1) and colistin (MIC 256 µg ml^–1) as well as enhanced LL‐37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA‐mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high‐level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross‐resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High‐level resistance to AMPs may contribute to the pathogenesis of US_NmUC.     

Adaptations of skin peptide defences and possible response to the amphibian chytrid fungus in populations of Australian green‐eyed treefrogs,Litoria genimaculata

Aim Rapidly evolving pathogens may exert diversifying selection on genes involved in host immune defence including those encoding antimicrobial peptides (AMPs). Amphibian skin peptides are one important defence against chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). We examined the population‐level variation in this innate immune defence to understand its relationship with disease dynamics. Location Queensland, Australia. Methods We examined skin peptide defences in five geographically distinct populations of Australian green‐eyed treefrogs, Litoria genimaculata. Skin peptide samples were collected from 52 frogs from three upland populations that previously declined as chytridiomycosis emerged, but subsequently recovered, and from 34 frogs in two lowland populations that did not decline. Historical samples of skin peptides preceding Bd emergence were not available from any population. Results In general, lowland populations had more effective peptide defences than upland populations. Peptide profiles were similar among populations, although relative amounts of peptides expressed differed significantly among populations and were more variable in the uplands. Infected frogs in upland populations carried a significantly higher infection burden compared to lowland populations. The presence of effective AMPs in the skin of L. genimaculata does not eliminate infection; however, more effective peptide defences may limit infection intensity and the progression of disease. Main conclusions The population bottleneck in upland populations caused by chytridiomycosis emergence did not appear to produce responses to selection for more effective peptide defences against chytridiomycosis compared to lowland populations of L. genimaculata. This does not exclude the possibility that current peptide defences have adapted in response to disease emergence. A suggestive (P < 0.10) interaction between infection status and population indicates that in lowland populations, infected individuals tend to be those with lower relative intensities of AMPs, whereas in the upland populations, infected and uninfected individuals are similar. Thus, both the AMPs and the environment may act to mediate resistance to Bd infection.     

Extremely abundant antimicrobial peptides existed in the skins of nine kinds of Chinese odorous frogs

Peptide agents are regarded as hopeful candidates to solve life-threatening resistance of pathogenic microorganisms to classic antibiotics due to their unique action mechanisms. Peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) from amphibian skin secretions can provide a large amount of structure-functional information to design peptide antibiotics with therapeutic potential. In the present study, we identified a large number of AMPs from the skins of nine kinds of Chinese odorous frogs. Eighty AMPs were purified from three different odorous frogs and confirmed by peptidomic analysis. Our results indicated that post-translational modification of AMPs rarely happened in odorous frogs. cDNAs encoding precursors of 728 AMPs, including all the precursors of the confirmed 80 native peptides, were cloned from the constructed AMP cDNA libraries of nine Chinese odorous frogs. On the basis of the sequence similarity of deduced mature peptides, these 728 AMPs were grouped into 97 different families in which 71 novel families were identified. Out of these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species. Our results revealed that identical AMPs were widely distributed in odorous frogs; 49 presently identified AMPs could find their identical molecules in different amphibian species. Purified peptides showed strong antimicrobial activities against 4 tested microbe strains. Twenty-three deduced peptides were synthesized and their bioactivities, including antimicrobial, antioxidant, hemolytic, immunomodulatory and insulin-releasing activities, were evaluated. Our findings demonstrate the extreme diversity of AMPs in amphibian skins and provide plenty of templates to develop novel peptide antibiotics.