“Shotgun” cloning of skin peptide precursors from skin secretion-derived cDNA libraries of the Fujian large-headed frog, Limnonectes fujianensis

Amphibian skin secretions are rich sources of biologically-active peptides and several studies involving molecular cloning of their biosynthetic precursors have revealed that many exhibit highly-conserved domain architectures with an associated high degree of primary structural conservation of the signal peptides. This conservation of primary structure is reflected at the level of nucleotide sequence — a finding that has permitted our group to design primers to these sites facilitating “shotgun” cloning using cDNA libraries from uninvestigated species. Here we describe the results of such an approach using a skin secretion-derived cDNA library from the Fujian large-headed frog, Limnonectes fujianensis, a completely unstudied species. In over 50 clones studied by this approach, 12 were found to encode peptides of different primary structure. Representatives of 5 different families of antimicrobial peptides derived from the skins of ranid frogs were found and these were brevinin-1 (n = 3), the ranatuerin-2 (n = 3), esculentin-2 (n = 1), temporin (n = 1) and chensinin (n = 1). Three clones encoded peptides that were novel with no homologues present in contemporary on-line databases. These included two related 16-mer peptides, named peptides SC-16a and b, and an unrelated 24-mer, named peptide AG-24. Preliminary biological characterisation of SC-16a has demonstrated an antimicrobial activity against Gram-negative bacteria with a minimal inhibitory concentration of 35 µM with no observable haemolysis up to 200 µM. This finding may suggest that this peptide represents a novel class of antimicrobial with little effect on eukaryotic membranes.

Kasstasin: A novel potent vasoconstrictor peptide from the skin secretion of the African red-legged running frog, Kassina maculata

Amphibian skin secretions are established sources of bioactive peptides. Here we describe the isolation, structural and pharmacological characterisation of a novel vasoconstrictor peptide from the skin secretion of the African hyperoliid frog, Kassina maculata, which exhibits no structural similarity to any known class of amphibian skin peptide. The peptide consists of 21 amino acid residues, FIKELLPHLSGIIDSVANAIK, and is C-terminally amidated. The provisional structure was obtained by MS/MS fragmentation using an Orbitrap mass spectrometer and L/I ambiguities were resolved following molecular cloning of biosynthetic precursor-encoding cDNA. A synthetic replicate of the peptide was found to possess weak antimicrobial and haemolytic activities but was exceptionally effective in constricting the smooth muscle of rat tail artery (EC50 of 25pM). In reflection of its exceptional potency in constricting rat arterial smooth muscle, the peptide was named kasstasin, a derivation of Kassina and “stasis” (stoppage of flow). These data illustrate the continuing potential of amphibian skin secretions to provide novel natural peptide templates for biological evaluation.

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu?Pro at position 2 and Phe?Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20µM and 150µM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His?Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5µM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His?Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40µm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.

A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.

Antimicrobial peptides and alytesin are co-secreted from the venom of the Midwife toad, Alytes maurus (Alytidae, Anura): Implications for the evolution of frog skin defensive secretions

The skin secretions of frogs and toads (Anura) have long been a known source of a vast abundance of bioactive substances. In the past decade, transcriptome data of the granular glands of anuran skin has given new impetus to investigations of the putative constituent peptides. Alytes obstetricans was recently investigated and novel peptides with antimicrobial activity were isolated and functionally characterised. However, genetic data for the evolutionarily ancient lineage to which Alytes belongs (midwife toads; Alytidae) remains unavailable.

Here we present the first such genetic data for Alytidae, derived via the granular gland transcriptome of a closely-related species of midwife toad, Alytes maurus. First, we present nucleotide sequences of the entire peptide precursors for four novel antimicrobial peptides (AMPs). The two precursors resemble those from Bombinatoridae in both their structural architecture and amino acid sequence. Each precursor comprises two AMPs as tandem repeats, with a member of the alyteserin-1 family (alyteserin-1Ma: GFKEVLKADLGSLVKGIAAHVAN-NH2 or alyteserin-1Mb: GFKEVLKAGLGSLVKGIPAHVAN-NH2) followed by its corresponding member from the alyteserin-2 family (alyteserin-2Ma: FIGKLISAASGLLSHL-NH2 or alyteserin-2Mb: ILGAIIPLVSGLLSHL-NH2). Synthetic replicates of the four AMPs possessed minimal inhibitory concentrations (MICs) ranging from 9.5 to 300 µM, with the most potent being alyteserin-2Ma. Second, we also cloned the cDNA encoding an alytesin precursor, with the active alytesin exhibiting high sequence identity to bombesin-related peptides from other frogs. All putative mature peptide sequences were confirmed to be present in the skin secretion via LC/MS.

The close structural resemblance of the alyteserin genes that we isolated for A. maurus with those of Bombina provide independent molecular evidence for a close evolutionary relationship between these genera as well as more support for the convergent evolution of the AMP system within anurans. In contrast to the more evolutionarily conserved nature of neuropeptides (including alytesin, which we also isolated), the more variable nature of the AMP system together with the sporadic distribution of AMPs among anuran amphibians fuels in part our hypothesis that the latter system was co-opted secondarily to fulfil a function in the innate immune system, having originally evolved for defence against potential macropredators.

Defense peptides secreted by helminth pathogens: antimicrobial and/or immunomodulator molecules?

Host defense peptides (HDPs) are an evolutionarily conserved component of the innate immune response found in all living species. They possess antimicrobial activities against a broad range of organisms including bacteria, fungi, eukaryotic parasites, and viruses. HDPs also have the ability to enhance immune responses by acting as immunomodulators. We discovered a new family of HDPs derived from pathogenic helminth (worms) that cause enormous disease in animals and humans worldwide. The discovery of these peptides was based on their similar biochemical and functional characteristics to the human defense peptide LL-37. We propose that these new peptides modulate the immune response via molecular mimicry of mammalian HDPs thus providing a mechanism behind the anti-inflammatory properties of helminth infections.

Senegalin: a novel antimicrobial/myotropic hexadecapeptide from the skin secretion of the African running frog, Kassina senegalensis

Amphibian skin is a rich and unique source of novel bioactive peptides most of which are endowed with either antimicrobial or pharmacological properties. Here we report the identification and structural characterization of a novel peptide, named senegalin, which possesses both activities. Senegalin is a hexadecapeptide amide (FLPFLIPALTSLISSL-NH2) of unique primary structure found in the skin secretion of the African running frog, Kassina senegalensis. The structure of the biosynthetic precursor of senegalin, deduced from cloned skin cDNA, consists of 76 amino acid residues and displays the typical domain organization of an amphibian skin peptide precursor. Both natural senegalin and its synthetic replicate
displayed antimicrobial and myotropic activities. Senegalin was active against Staphylococcus aureus (MIC 50µM) and Candida albicans (MIC 150µM) but was nonhaemolytic at concentrations up to and including 150µM. In contrast, senegalin induced a dose-dependent contraction of rat urinary bladder smooth muscle (EC50 2.9nM) and a dosedependent relaxation of rat tail artery smooth muscle (EC50 37.7nM). Senegalin thus represents a prototype biologically-active amphibian skin peptide and illustrates the fact thatamphibian skin secretion peptidomes continue to be unique sources of such molecules.

Molecular evolution of proadrenomedullin N-terminal 20 peptide (PAMP):evidence for gene co-option

Posttranslational processing of proadrenomedullin generates two biologically active peptides, adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Sequence comparison of homologous proadrenomedullin genes in vertebrate evolution shows a high degree of stability in the reading frame for AM, whereas PAMP sequence changes rapidly. Here we investigate the functional significance of PAMP phylogenetic variation studying two of PAMP's better characterized physiological activities, angiogenic potential and antimicrobial capability, with synthetic peptides carrying the predicted sequence for human, mouse, chicken, and fish PAMP. All tested peptides induced angiogenesis when compared with untreated controls, but chicken and fish PAMP, which lack terminal amidation, were apparently less angiogenic than their human and mouse homologs. Confirming the role of amidation in angiogenesis, Gly-extended and free acid variants of human PAMP produced responses similar to the natural nonamidated peptides. In contrast, antimicrobial activity was restricted to human PAMP, indicating that this function may have been acquired at a late time during the evolution of PAMP. Interestingly, free acid human PAMP retained antimicrobial activity whereas the Gly-extended form did not. This fact may reflect the need for maintaining a tightly defined structural conformation in the pore-forming mechanism proposed for these antimicrobial agents. The evolution of PAMP provides an example of an angiogenic peptide that developed antimicrobial capabilities without losing its original function.

AcT-2: A Novel Myotropic and Antimicrobial Type 2 Tryptophyllin from the Skin Secretion of the Central American Red-Eyed Leaf Frog, Agalychnis callidryas

Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure—GMRPPWF-NH2—was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μM). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Balteatide: A Novel Antimicrobial Decapeptide from the Skin Secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea

The skin secretions of Neotropical phyllomedusine leaf frogs have proven to be a rich source of biologically-active peptides, including antimicrobials. The major families of antimicrobial peptides (AMPs) reported are the dermaseptins and phylloseptins and the minor families, the dermatoxins, phylloxins, plasticins, distinctins and the medusins. Here, we report a novel AMP of 10 amino acid residues (LRPAILVRIKamide), named balteatide, from the skin secretion of wild Peruvian purple-sided leaf frogs, Phyllomedusa baltea. Balteatide was found to exhibit a 90% sequence identity with sauvatide, a potent myotropic peptide from the skin secretion of Phyllomedusa sauvagei. However, despite both peptides exhibiting only a single amino acid difference (I/T at position 9), sauvatide is devoid of antimicrobial activity and balteatide is devoid of myotropic activity. Balteatide was found to have differential activity against the Gram-positive bacterium, Staphylococcus aureus, the Gram-negative bacterium, Escherichia coli and the yeast, Candida albicans, and unusually for phyllomedusine frog skin AMPs, was most potent (MIC 32 mg/L) against the yeast. Balteatide was also devoid of haemolytic activity up to concentrations of 512 mg/L. Phyllomedusine frog skin secretions thus continue to provide novel AMPs, some of which may provide templates for the rational design of new classes of anti-infective therapeutics.

Feleucin-BO1: A Novel Antimicrobial Non-Apeptide Amide from the Skin Secretion of the Toad, Bombina orientalis, and Design of a Potent Broad-Spectrum Synthetic Analogue, Feleucin-K3

Feleucins-BV1 and -BV2 are recently-described prototypes of a novel antimicrobial nonapeptide (AMP) family identified in the skin secretion of the bombinid toad, Bombina variegata. They are encoded on different precursors that also encode a novel bombinin. Here we describe the identification of feleucin-BO1 (FLGLLGSLLamide) which is co-encoded with a different novel bombinin, named feleucin precursor-associated bombinin (FPA-bombinin-BO), from the skin secretion of Bombina orientalis. Synthetic feleucin-BO1 displayed activity against a reference Gram-positive bacterium. Staphylococcus aureus (MIC 34 μM) but was inactive (> 250 μM) against the Gram-negative bacterium, Escherichia coli, and the yeast, Candida albicans. This pattern of activity was similar to that of the prototypes. Design and synthesis of a cationicity-enhanced analogue, feleucin-K3 (F-K3), in which the amino acid residues at positions 3 (G), 6 (G) and 7 (S) of feleucin-BO1 were substituted with Lys (K) residues, resulted in a peptide with significantly-enhanced potency and spectrum of activity. The MICs of F-K3 against the reference microorganisms were 7 μM (S. aureus), 14 μM (E. coli) and 7 μM (C. albicans). These data indicate that the skin secretions of amphibians can continue to provide novel peptide templates for the rational design of analogues with possible therapeutic utility.

Control of cutaneous antimicrobial peptides by vitamin D3

Constant exposure to a wide variety of microbial pathogens represents a major challenge for our skin. Antimicrobial peptides (AMPs) are mediators of cutaneous innate immunity and protect primarily against microbial infections. Cathelicidins were among the first AMPs identified in human skin and recent evidence suggests that they exert a dual role in innate immune defense: At first, due to their antimicrobial activity they kill pathogens directly. In addition, these peptides initiate a potent host response to infection resulting in cytokine release, inflammation and a cellular response. Disturbed cathelicidin expression and function was observed in several common inflammatory skin diseases, such as psoriasis where cathelicidin peptide converts inert self-DNA and self-RNA into an autoimmune stimulus. In atopic dermatitis decreased levels of cathelicidin facilitating microbial superinfections have been discussed. Furthermore, abnormally processed cathelicidin peptides induce inflammation and a vascular response in rosacea. Until recently, the molecular mechanisms underlying cathelicidin regulation were unknown. Recently, the vitamin D3 pathway was identified as the major regulator of cathelicidin expression. Consequently, vitamin D3 entered the spotlight as an immune modulator with impact on both innate and adaptive immunity. Therapies targeting vitamin D3 signaling may provide new approaches for infectious and inflammatory skin diseases by affecting both innate and adaptive immune functions.

Evolution of Antimicrobial Peptides to Self-Assembled Peptides for Biomaterial Applications

Biomaterial-related infections are a persistent burden on patient health, recovery, mortality and healthcare budgets. Self-assembled antimicrobial peptides have evolved from the area of antimicrobial peptides. Peptides serve as important weapons in nature, and increasingly medicine, for combating microbial infection and biofilms. Self-assembled peptides harness a “bottom-up” approach, whereby the primary peptide sequence may be modified with natural and unnatural amino acids to produce an inherently antimicrobial hydrogel. Gelation may be tailored to occur in the presence of physiological and infective indicators (e.g. pH, enzymes) and therefore allow local, targeted antimicrobial therapy at the site of infection. Peptides demonstrate inherent biocompatibility, antimicrobial activity, biodegradability and numerous functional groups. They are therefore prime candidates for the production of polymeric molecules that have the potential to be conjugated to biomaterials with precision. Non-native chemistries and functional groups are easily incorporated into the peptide backbone allowing peptide hydrogels to be tailored to specific functional requirements. This article reviews an area of increasing interest, namely self-assembled peptides and their potential therapeutic applications as innovative hydrogels and biomaterials in the prevention of biofilm-related infection.

Direct and indirect antimicrobial activities of neuropeptides and their therapeutic potential

As global resistance to conventional antibiotics rises we need to develop new strategies to develop future novel therapeutics. In our quest to design novel anti-infectives and antimicrobials it is of interest to investigate host-pathogen interactions and learn from the complexity of host defense strategies that have evolved over millennia. A myriad of host defense molecules are now known to play a role in protection against human infection. However, the interaction between host and pathogen is recognized to be a multifaceted one, involving countless host proteins, including several families of peptides. The regulation of infection and inflammation by multiple peptide families may represent an evolutionary failsafe in terms of functional degeneracy and emphasizes the significance of host defense in survival. One such family is the neuropeptides (NPs), which are conventionally defined as peptide neurotransmitters but have recently been shown to be pleiotropic molecules that are integral components of the nervous and immune systems. In this review we address the antimicrobial and anti-infective effects of NPs both in vitro and in vivo and discuss their potential therapeutic usefulness in overcoming infectious diseases. With improved understanding of the efficacy of NPs, these molecules could become an important part of our arsenal of weapons in the treatment of infection and inflammation. It is envisaged that targeted therapy approaches that selectively exploit the anti-infective, antimicrobial and immunomodulatory properties of NPs could become useful adjuncts to our current therapeutic modalities. © 2012 Bentham Science Publishers.

Anti-biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device-related pathogens

The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug-resistant pathogens. Biofilm-forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra-peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram-positive medical device-related pathogens. 3-(4-Hydroxyphenyl)propionic)-Orn-Orn-Trp-Trp-NH2 displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24-h biofilms at MBEC with 6-h exposure. Reduced cell cytotoxicity, relative to Gram-positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes).

Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost-effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied.