Sequence polymorphisms of PR39 cathelicidins and extensive copy variations in commercial pig breeds.

Copy number polymorphisms (CNPs) of antimicrobial peptides (AMPs) in livestock can influence the innate immune response of individuals. We conducted a high-resolution analysis of the genomic variations of porcine cathelicidin PR39 using cloned PR39 amplicons corresponding to the 5' untranslated region (UTR) to 3' UTR from four individuals of three different pig breeds. We identified 15 different sequences corresponding to 9 different coding domain sequences (CDSs), encoding 7 different protein sequences consisting of 3 functional and 4 non-functional forms. Subsequently, we developed a PR39 CNP typing method using real-time polymerase chain reaction (PCR) and analyzed the PR39 copy numbers from 44 pigs of six breeds. Significant variations in PR39 copies ranging from 2 to 10 copies, with a mean copy number of 5, were observed among all commercial breeds, except the wild boar. Among the different breeds, the PR39 copy number was highest (10) in Korean native pigs. Gene expression analysis showed that PR39 expression was correlated with the copy number. Moreover, the comparative analysis of the cathelicidin cluster-containing region among eight mammalian species showed the complete evolutionary conservation of the region, except for differences in the degree of cathelicidin expansion in each species. Therefore, characterization of CNPs in AMP genes could aid in improving the genetic potential of innate immune responses in livestock animals.

Diversity, immunoregulatory action and structure-activity relationship of green sea turtle cathelicidins.

Cathelicidins are a family of gene-encoded immune effectors in vertebrate innate immunity. Here, we reported the diversity and biological activity of cathelicidins in green sea turtle, a marine reptile species known for long lifespan and disease resistance. Four novel cathelicidins (Cm-CATH1-4) were identified from green sea turtle. All of them, especially Cm-CATH2, exhibited potent, broad-spectrum and rapid bactericidal and anti-biofilm activities by inducing the disruption of cell membrane integrity. Additionally, Cm-CATH2 effectively induced the macrophages/monocytes and neutrophils trafficking to the infection site, and inhibited the LPS-induced production of inflammatory cytokines, by blocking TLR4/MD2 complex and the downstream signaling pathway activation. In mouse peritonitis and pneumonia models, Cm-CATH2 exhibited evident protection against drug-resistant bacterial infections. Taken together, the diverse structures and functions of Cm-CATHs indicated their pleiotropic role in innate immunity of green sea turtle, and the potent antimicrobial, anti-biofilm and immunomodulatory properties make them ideal candidates for the development of novel anti-infective drugs.

Functions of Antimicrobial Peptides in Vertebrates.

OBJECTIVE: The aim of this review is to examine the multiple activities of antimicrobial peptides (AMPs) in vertebrates. CONTENT: The largest AMP families are the cathelicidins and defensins, but several peptides derived from bigger proteins have also been reported. Cathelicidins are characterized by a conserved Nterminal pro-region and a variable region that encodes the C-terminal mature peptide. The ß-defensins comprise a large family of AMPs that have diversified their functions, apparently without losing their antimicrobial activity. Cathelicidins and ß-defensins are present in all vertebrates studied so far; α- defensins are present in mammals, while θ-defensins are only present in some non-human primates. The AMPs are regulated by posttranslational modifications that mainly include proteolysis, amidation, ADP-ribosylation, glycosylation and phosphorylation. In addition to their antimicrobial effects, AMPs show activity against viral particles and interfere in different steps of virus replication. Moreover, AMPs may both promote and inhibit cancer growth: several vertebrate AMPs kill cancer cells, and some tumors grow in an environment wherein the expression of ß-defensins is reduced; however, human cathelicidin and some ß-defensins are overexpressed in several types of cancer and are correlated with tumor growth. AMPs are part of the complex network of cells and molecules that forms the vertebrate innate defense system and they induce adaptive responses. In addition, they participate in sperm maturation and male reproduction. CONCLUSION: AMPs are multifunctional peptides that participate in immune responses, wound healing, angiogenesis, toxin neutralization, iron metabolism, male reproduction, among other functions. However, AMPs may also contribute to excessive inflammation and tumorigenesis.

Diversity, Antimicrobial Action and Structure-Activity Relationship of Buffalo Cathelicidins.

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.

Brief Note :Variability in the cathelicidin 6 (CATHL-6) gene in Tianzhu white yak from Tibetan area in China.

Cathelicidins are a major family of antimicrobial peptides (AMPs), an important component of innate immune system, playing a critical role in host defense and disease resistance in virtually all living species. Polymorphism and functional studies on cathelicidin of Tianzhu white yak contribute to understanding the specific innate immune mechanism in animals living at high altitudes in comparison to cattle and domesticated white yak. Thirty-six individuals of Tianzhu white yak, originating from the area of three ecotypes (Gansu in China), were investigated. The total length of the aligned Yak cathelicidin 6 (CATHL-6) sequences was 1923 bp, including six single nucleotide polymorphisms and one indel. Ten haplotypes were identified, and phylogenetic analyses resolved those 10 haplotypes in two clusters. The results indicate that the white yak originated from two domestication sites. In addition, lack of significant pairwise difference between sequences (Tajima's D = 0.92865, P > 0.10) in the CATHL-6 region indicates absence of population size expansion in current white yak population.

Identification and polymorphism discovery of the cathelicidins, Lf-CATHs in ranid amphibian (Limnonectes fragilis).

To protect themselves against the invasion of microorganisms, amphibians, especially the Rana frogs, are possibly equipped with complex combinations of antimicrobial peptides (AMPs). The two major AMP families, ranid skin secretion AMPs and cathelicidins might together constitute the host innate immune system of amphibians. Cathelicidins are a group of cationic peptides found in leukocytes and epithelial cells, and they play a central role in the early innate immune defense found in virtually all species of mammals. However, they have rarely been reported from amphibians. Here, we report the identification and discovery of polymorphism cathelicidins in Limnonectes fragilis. The expression profile indicated high cathelicidin transcript levels in frog spleen, liver and kidney, but lower levels in lung, skin and stomach. According to the amphibian's unique proteolytic pattern, R125 and L121 of the prepropeptides are predicted to be the processing positions for protease to generate the mature peptides, Lf-CATH1 and -2, respectively. Both consist of 30 amino acid residues, of which two were cysteines positionally conserved among a few known amphibian cathelicidins. Homology modeling analysis revealed that Lf-CATH1 and -2 adopt a tertiary structure with a mostly α helix that is representative of small cationic cathelicidin family peptides. Recombinant Lf-CATH1 (rLf-CATH1) was produced in Escherichia coli. Synthetic Lf-CATH1 and -2 displayed potent antimicrobial activities in vitro against a broad spectrum of microorganisms, including standard and clinically isolated drug-resistant strains, while showing neglectable hemolysis and cytotoxicities.

Identification and characterization of a novel cathelicidin from ayu, Plecoglossus altivelis.

Cathelicidins, one family of antimicrobial peptides, play important roles against infections in animals. In this study, a cDNA sequence coding for cathelicidin was cloned from constructed liver cDNA library of ayu, Plecoglossus altivelis. The deduced ayu cathelicidin (aCATH) has a 20 amino acid residue signal peptide, a conserved cathelin domain of 110 amino acid residues and a mature antimicrobial peptide of 61 amino acid residues. Sequence comparison and phylogenetic tree analysis confirmed aCATH as a distinct member of fish cathelicidins. Real-time quantitative PCR revealed that the aCATH transcripts dramatically increased in various tissues after bacterial infection. Subsequently, aCATH was prokaryotic expressed and purified. Western blot and mass spectrometry revealed that aCATH was cleaved at residue Ile130-Arg131 by human neutrophil elastase to release the mature antimicrobial peptide. The mature peptide of aCATH was chemically synthesized and exhibited potent antimicrobial activity. Thus, aCATH may play an important role in the innate immunity of ayu, and this work enriches our knowledge in fish antimicrobial peptides.

Differential antimicrobial peptide gene expression patterns during early chicken embryological development.

The adaptive immune system is not completely developed when chickens hatch, so the innate immune system has evolved a range of mechanisms to deal with early pathogenic assault. Avian beta-defensins (AvBDs) and cathelicidins (CTHLs) are two major sub-classes of antimicrobial peptides (AMPs) with a fundamental role in both innate and adaptive immune responses. In this study, we demonstrate distinct expression patterns of innate immune genes including - Toll-like receptors (TLRs) (TLR2, TLR15 and TLR21, but not TLR4), the complete repertoire of AvBDs, CTHLs and both pro- and anti-inflammatory cytokines (IL1B, IL8, IFNG and IL10) during early chicken embryonic development. AvBD9 was significantly increased by over 150 fold at day 9; and AvBD10 was increased by over 100 fold at day 12 in the abdomen of the embryo, relative to day 3 expression levels (P<0.01). In contrast, AvBD14 was preferentially expressed in the head of the embryo. This is the first study to demonstrate differential patterns of AMP gene expression in the sterile environment of the developing embryo. Our results propose novel roles for AMPs during development and reveal the innate preparedness of developing embryos for pathogenic assault in ovo, or post-hatching.

Lipopolysaccharide bound structures of the active fragments of fowlicidin-1, a cathelicidin family of antimicrobial and antiendotoxic peptide from chicken, determined by transferred nuclear Overhauser effect spectroscopy.

Cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. The C-terminal part of the cathelicidins is bestowed with antimicrobial and lipopolysaccharide (LPS) neutralizing activities. In this work, we repot high resolution solution structures of two nontoxic active fragments, residues 1-16 or RG16 and residues 8-26 or LK19, of fowlicidin-1, a cathelicidin family of peptide from chicken, as a complex with LPS using two-dimensional transferred nuclear Overhauser effect (Tr-NOE) spectroscopy. Both peptides are highly flexible and do not assume any preferred conformations in their free states. Upon complexation with endotoxin or LPS, peptides undergo structural transitions towards folded conformations. Structure calculations reveal that the LK19 peptide adopts a well defined helical structure with a bend at the middle. By contrast, the first seven amino acids of RG16 are found to be flexible followed by a helical conformation for the residues L8-A15. In addition, a truncated version of LK19 encompassing residues A15-K26 or AK12 displays an amphipathic helical structure in LPS. Saturation transfer difference (STD) NMR studies demonstrate that all peptides, RG16, LK19, and AK12, are in close proximity with LPS, whereby the aromatic residues showed the strongest STD effects. Fluorescence studies with fluorescein isothiocyanate (FITC) labeled LPS in the presence of full-length fowlicidin-1, LK19, RG16, and AK12 indicated that LPS-neutralization property of these peptides may result from plausible dissociation of LPS aggregates. The helical structures of peptide fragments derived from fowlicidin-1 in LPS could be utilized to develop nontoxic antiendotoxic compounds.

Identification and characterization of novel reptile cathelicidins from elapid snakes.

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576bp and coded for 191 amino acid residue protein precursors. Each of the deduced elapid cathelicidin has a 22 amino acid residue signal peptide, a conserved cathelin domain of 135 amino acid residues and a mature antimicrobial peptide of 34 amino acid residues. Unlike the highly divergent cathelicidins in mammals, the nucleotide and deduced protein sequences of the three cloned elapid cathelicidins were remarkably conserved. All the elapid mature cathelicidins were predicted to be cleaved at Valine157 by elastase. OH-CATH, the deduced mature cathelicidin from king cobra, was chemically synthesized and it showed strong antibacterial activity against various bacteria with minimal inhibitory concentration of 1-20microg/ml in the presence of 1% NaCl. Meanwhile, the synthetic peptide showed no haemolytic activity toward human red blood cells even at a high dose of 200microg/ml. Phylogenetic analysis of cathelicidins from vertebrate suggested that elapid and viperid cathelicidins were grouped together in the tree. Snake cathelicidins were evolutionary closely related to the neutrophilic granule proteins (NGPs) from mouse, rat and rabbit. Snake cathelicidins also showed a close relationship with avian fowlicidins (1-3) and chicken myeloid antimicrobial peptide 27. Elapid cathelicidins might be used as models for the development of novel therapeutic drugs.