Family Of Antimicrobial Peptides Research Articles

Molecular Insights into the Role of Cysteine-Rich Peptides in Induced Resistance to Fusarium oxysporum Infection in Tomato Based on Transcriptome Profiling.

Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.

A nematode-derived, mitochondrial stress signaling-regulated peptide exhibits broad antibacterial activity.

ABSTRACTA dramatic rise of infections with antibiotic-resistant bacterial pathogens continues to challenge the healthcare field due to the lack of effective treatment regimes. As such, there is an urgent need to develop new antimicrobial agents that can combat these multidrug-resistant superbugs. Mitochondria are central regulators of metabolism and other cellular functions, including the regulation of innate immunity pathways involved in the defense against infection. The mitochondrial unfolded protein response (UPRmt) is a stress-activated pathway that mitigates mitochondrial dysfunction through the regulation of genes that promote recovery of the organelle. In the model organism Caenorhabditis elegans, the UPRmt also mediates an antibacterial defense program that combats pathogen infection, which promotes host survival. We sought to identify and characterize antimicrobial effectors that are regulated during the UPRmt. From our search, we discovered that the antimicrobial peptide CNC-4 is upregulated during this stress response. CNC-4 belongs to the caenacin family of antimicrobial peptides, which are predominantly found in nematodes and are known to have anti-fungal properties. Here, we find that CNC-4 also possesses potent antimicrobial activity against a spectrum of bacterial species and report on its characterization.

Porcine Myeloid Antimicrobial Peptides: A Review of the Activity and Latest Advances.

Traditional antibiotics have made great contributions to human health and animal husbandry since the discovery of penicillin in 1928, but bacterial resistance and drug residues are growing threats to global public health due to the long-term uncontrolled application of antibiotics. There is a critical need to develop new antimicrobial drugs to replace antibiotics. Antimicrobial peptides (AMPs) are distributed in all kingdoms of life, presenting activity against pathogens as well as anticancer, anti-inflammatory, and immunomodulatory activities; consequently, they have prospects as new potential alternatives to antibiotics. Porcine myeloid antimicrobial peptides (PMAPs), the porcine cathelicidin family of AMPs, have been reported in the literature in recent years. PMAPs have become an important research topic due to their strong antibacterial activity. This review focuses on the universal trends in the biochemical parameters, structural characteristics and biological activities of PMAPs.

Molecular characterization of cathelicidin in tiger frog (Hoplobatrachus rugulosus): Antimicrobial activity and immunomodulatory activity

Cathelicidins are an important antimicrobial peptide family and are expressed in many different vertebrates. They play an important role in the innate immune system of the host. However, amphibian cathelicidins are poorly understood. In this study, the cDNA of the cathelicidin gene was obtained from the skin transcriptome of tiger frog (Hoplobatrachus rugulosus). The predicted amino acid sequence of tiger frog cathelicidin (HR-CATH) comprises a signal peptide, a cathelin domain, and a mature peptide. The HR-CATH amino acid sequence alignment with other frog cathelicidins showed that the functional mature peptide is highly variable in amphibians, whereas the cathelin domain is conserved. A phylogenetic tree analysis showed that HR-CATH is most closely related to cathelicidin-NV from Nanorana ventripunctata. HR-CATH was chemically synthesized and its in vitro activity was determined. It had high antibacterial activity against Vibrio parahaemolyticus, Staphylococcus aureus, and the pathogenic bacterium Aeromonas hydrophila. HR-CATH damaged the cell membrane integrity of A. hydrophila according to a lactate dehydrogenase release assay and was able to hydrolyze the genomic DNA from A. hydrophila in a dose-dependent manner. Furthermore, in RAW264.7 cells (mouse leukemic monocyte/macrophage cell line), HR-CATH induced chemotaxis and enhanced respiratory burst. Our study shows that amphibian cathelicidin has antimicrobial activity and an immunomodulatory effect on immune cells.

Barbel steed (Hemibarbus labeo) NK-lysin protects against Aeromonas hydrophila infection via immunomodulatory activity

NK-lysins (NKLs) are a family of multifunctional antimicrobial peptides that have activity against various microorganisms. However, the immunomodulatory activity of NKL in fish remains unclear. In this study, the cDNA sequence of barbel steed (Hemibarbus labeo) NKL gene was cloned. Barbel steed NKL amino acid sequence comprised a signal peptide and a mature peptide. The saposin B domain in the mature peptide has six conserved cysteines that form three disulfide bonds. Phylogenetic analysis showed that the barbel steed NKL was most closely related to that of the common carp (Cyprinus carpio) NKL. Differential expression analysis showed that the barbel steed NKL gene was expressed in all tested tissues, with the highest expression in the spleen. In response to Aeromonas hydrophila infection, NKL was significantly upregulated in the liver, spleen, head kidney, and gill. The barbel steed NKL showed strong antibacterial activity against Vibrio parahaemolyticus, V. alginolyticus, V. vulnificus, and Listeria monocytogenes. However, NKL had no antibacterial activity against the pathogenic bacteria A. hydrophila. Lactate dehydrogenase release assays showed that NKL damaged the V. parahaemolyticus cell membrane. NKL significantly increased barbel steed survival rate after A. hydrophila infection and upregulated IL-1β and TNF-α expression in the spleen and head kidney. NKL induced monocyte/macrophage chemotaxis and enhanced the respiratory burst and proinflammatory cytokine expression. Our study shows that fish NKL exhibits immunomodulatory effects and protects the host from pathogenic infections independent of direct bacterial clearance.

Hepcidin Increases Cytokines in Alzheimer's Disease and Down's Syndrome Dementia: Implication of Impaired Iron Homeostasis in Neuroinflammation.

The liver-derived hormone hepcidin, a member of the defensin family of antimicrobial peptides, plays an important role in host defense and innate immunity due to its broad antibacterial and antiviral properties. Ferritin, an iron storage protein is often associated with iron deficiency, hypoferritinemia, hypoxia, and immune complications, which are all significant concerns for systemic infection in Alzheimer’s disease (AD) and Down’s syndrome (DS) dementia. Serum and post-mortem brain samples were collected from AD, DS and age-matched control subjects. Serum samples were analyzed with ELISA for ferritin, hepcidin and IL-6. Additionally, post-mortem brain sections were assessed by immunohistochemistry for iron-related and inflammatory proteins. A significant increase in serum hepcidin levels was found in DS, compared to controls and AD subjects (p < 0.0001). Hepcidin protein was visible in the epithelial cells of choroid plexus, meningeal macrophages and in the astrocytes close to the endothelium of blood vessels. Hepcidin co-localized with IL-6, indicating its anti-inflammatory properties. We found significant correlation between hypoferritinemia and elevated levels of serum hepcidin in AD and DS. Hepcidin can be transported via macrophages and the majority of the vesicular hepcidin enters the brain via a compromised blood brain barrier (BBB). Our findings provide further insight into the molecular implications of the altered iron metabolism in acute inflammation, and can aid towards the development of preventive strategies and novel treatments in the fight against neuroinflammation.

Aggregation and Its Influence on the Bioactivities of a Novel Antimicrobial Peptide, Temporin-PF, and Its Analogues.

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl2 and MgCl2 buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.

Overlapping Properties of the Short Membrane-Active Peptide BP100 With (i) Polycationic TAT and (ii) α-helical Magainin Family Peptides.

BP100 is a short, designer-made membrane-active peptide with multiple functionalities: antimicrobial, cell-penetrating, and fusogenic. Consisting of five lysines and 6 hydrophobic residues, BP100 was shown to bind to lipid bilayers as an amphipathic α-helix, but its mechanism of action remains unclear. With these features, BP100 embodies the characteristics of two distinctly different classes of membrane-active peptides, which have been studied in detail and where the mechanism of action is better understood. On the one hand, its amphiphilic helical structure is similar to the pore forming magainin family of antimicrobial peptides, though BP100 is much too short to span the membrane. On the other hand, its length and high charge density are reminiscent of the HIV-TAT family of cell penetrating peptides, for which inverted micelles have been postulated as translocation intermediates, amongst other mechanisms. Assays were performed to test the antimicrobial and hemolytic activity, the induced leakage and fusion of lipid vesicles, and cell uptake. From these results the functional profiles of BP100, HIV-TAT, and the magainin-like peptides magainin 2, PGLa, MSI-103, and MAP were determined and compared. It is observed that the activity of BP100 resembles most closely the much longer amphipathic α-helical magainin-like peptides, with high antimicrobial activity along with considerable fusogenic and hemolytic effects. In contrast, HIV-TAT shows almost no antimicrobial, fusogenic, or hemolytic effects. We conclude that the amphipathic helix of BP100 has a similar membrane-based activity as magainin-like peptides and may have a similar mechanism of action.

Structural and functional characterization of NDBP-4 family antimicrobial peptides from the scorpion Mesomexovis variegatus

Six peptides, belonging to the NDBP-4 family of scorpion antimicrobial peptides were structurally and functionally characterized. The sequence of the mature peptides VpCT1, VpCT2, VpCT3 and VpCT4 was inferred by transcriptomic analysis of the venom gland of the scorpion Mesomexovis variegatus. Analysis of their amino acid sequences revealed patterns that are also present in previously reported peptides that show differences in their hemolytic and antimicrobial activities in vitro. Two other variants, VpCT3W and VpCTConsensus were designed to evaluate the effect of sequence changes of interest on their structure and activity. The synthesized peptides were evaluated by circular dichroism to confirm their α-helical conformation in a folding promoting medium. The peptides were assayed on two Gram-positive and three Gram-negative bacterial strains, and on two yeast strains. They preferentially inhibited the growth of Staphylococcus aureus, were mostly ineffective on Pseudomonas aeruginosa, and moderately inhibited the growth of Candida yeasts. All six peptides exhibited hemolytic activity on human erythrocytes in the range of 4.8–83.7 μM. VpCT3W displayed increased hemolytic and anti-yeast activities, but showed no change in antibacterial activity, relative to its parental peptide, suggesting that Trp6 may potentiate the interaction of VpCT3 with eukaryotic cell membranes. VpCTConsensus showed broader and enhanced antimicrobial activity relative to several of the natural peptides. The results presented here contribute new information on the structure and function of NDBP-4 antimicrobial peptides and provides clues for the design of less hemolytic and more effective antimicrobial peptides.

Antimicrobial Bombinin-like Peptide 3 Selectively Recognizes and Inserts into Bacterial Biomimetic Bilayers in Multiple Steps.

Bombinins are a wide family of antimicrobial peptides from Xenopus skin. By sequence clustering, we highlighted at least three families named A, B, and H, which might exert antibacterial activity by different modes of action. In this work, we study bombinin-like peptide 3 (BLP-3) as a nonhemolytic representative of the quite unexplored class A due to its appealing activity toward WHO-priority-list bacteria such as Neisseria, Pseudomonas aeruginosa, and Staphylococcus aureus. A marked preference for cardiolipin and phosphatidylglycerol head groups, typically found in bacteria, is proven with biomimetic membranes studied by liquid and solid NMR and MD simulations. BLP-3 gets structured upon interaction and penetrates deeply into the bilayer in two steps involving a superficial insertion of key side chains and subsequent internalization. All along the pathway, a fundamental role is played by lysine residues in the conserved region 11-19, which act in synergy with other key residues.

Koala cathelicidin PhciCath5 has antimicrobial activity, including against Chlamydia pecorum.

Devastating fires in Australia over 2019–20 decimated native fauna and flora, including koalas. The resulting population bottleneck, combined with significant loss of habitat, increases the vulnerability of remaining koala populations to threats which include disease. Chlamydia is one disease which causes significant morbidity and mortality in koalas. The predominant pathogenic species, Chlamydia pecorum, causes severe ocular, urogenital and reproductive tract disease. In marsupials, including the koala, gene expansions of an antimicrobial peptide family known as cathelicidins have enabled protection of immunologically naïve pouch young during early development. We propose that koala cathelicidins are active against Chlamydia and other bacteria and fungi. Here we describe ten koala cathelicidins, five of which contained full length coding sequences that were widely expressed in tissues throughout the body. Focusing on these five, we investigate their antimicrobial activity against two koala C. pecorum isolates from distinct serovars; MarsBar and IPTaLE, as well as other bacteria and fungi. One cathelicidin, PhciCath5, inactivated C. pecorum IPTaLE and MarsBar elementary bodies and significantly reduced the number of inclusions compared to the control (p<0.0001). Despite evidence of cathelicidin expression within tissues known to be infected by Chlamydia, natural PhciCath5 concentrations may be inadequate in vivo to prevent or control C. pecorum infections in koalas. PhciCath5 also displayed antimicrobial activity against fungi and Gram negative and positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Electrostatic interactions likely drive PhciCath5 adherence to the pathogen cell membrane, followed by membrane permeabilisation leading to cell death. Activity against E. coli was reduced in the presence of 10% serum and 20% whole blood. Future modification of the PhciCath5 peptide to enhance activity, including in the presence of serum/blood, may provide a novel solution to Chlamydia infection in koalas and other species.

Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

BackgroundPlant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays.ResultsAtr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors.ConclusionsThis study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

Farfantepenaeus gene-encoded antimicrobial peptides: Identification, molecular characterization and gene expression in response to fungal infections

The aim of this study was to identify and characterize, at the molecular and transcriptional levels, sequences encoding the different members of the four families of shrimp antimicrobial peptides (AMPs) in species of the genus Farfantepenaeus. The identification of the AMP sequences was performed by in silico analysis as well as by molecular cloning and nucleotide sequencing. We identified all seven shrimp ALFs (ALF-A to ALF-G), both Type IIa and Type IIb crustins as well as two stylicins (STY1 and STY2) in Farfantepenaeus. Only two genes (PEN1/2 and PEN4) of the four-member penaeidin family (PEN1/2 to PEN5) were found and this is the first report of stylicins as well as of several additional members of ALFs, crustins and penaeidins in species of the genus Farfantepenaeus. All AMP genes have shown to be constitutively transcribed in the shrimp immune cells (hemocytes), except for ALF-G. Finally, the transcriptional profile of the different AMPs was assessed in the hemocytes of F. paulensis (pink shrimp) following an experimental infection with the opportunistic filamentous fungus Fusarium solani. We found that while the expression of ALF-B was induced at 24 h, the STY2 gene was down-regulated at 48 h post-challenge. These results provide evidence of the molecular diversity of AMPs from shrimp of the genus Farfantepenaeus in terms of sequences, biochemical properties and expression profiles in response to infectious diseases.

Cassava (Manihot esculenta) defensins: Prospection, structural analysis and tissue-specific expression under biotic/abiotic stresses

Defensins are a prominent family of antimicrobial peptides. They play sophisticated roles in the defense against pathogens in all living organisms, but few works address their expression under different conditions and plant tissues. The present work prospected defensins of Manihot esculenta Crantz, popularly known as cassava. Five defensin candidates (MeDefs) were retrieved from the genome sequences and characterized. Considering chromosome distribution, only MeDef1 and 2 occupy adjacent positions in the same chromosome arm. All 3D structures had antiparallel ß-sheets, an α-helix, and amphipathic residues distributed throughout the peptides with a predominance of cationic surface charge. MeDefs expression was validated by RT-qPCR, including two stress types (biotic: fungus Macrophomina pseudophaseolina, and abiotic: mechanical injury) and a combination of both stresses (fungus+injury) in three different tissues (root, stem, and leaf). For this purpose, ten reference genes (RGs) were tested, and three were chosen to characterize MeDef expression. MeDef3 was up-regulated at roots in all stress situations tested. MeDef1 and MeDef5 were induced in leaves under biotic and abiotic stresses, but not in both stress types simultaneously. Only MeDef2 was down-regulated in the stem tissue also with biotic/abiotic combined stresses. These results indicate that although defensins are known to be responsive to pathogen infection, they may act as preformed defense or, still, have tissue or stress specificities. Aspects of their structure, stability and evolution are also discussed.

Molecular Diversity and Evolution of Antimicrobial Peptides in Musca domestica

As a worldwide sanitary insect pest, the housefly Musca domestica can carry and transmit more than 100 human pathogens without suffering any illness itself, indicative of the high efficiency of its innate immune system. Antimicrobial peptides (AMPs) are the effectors of the innate immune system of multicellular organisms and establish the first line of defense to protect hosts from microbial infection. To explore the molecular diversity of the M. domestica AMPs and related evolutionary basis, we conducted a systematic survey of its full AMP components based on a combination of computational approaches. These components include the cysteine-containing peptides (MdDefensins, MdEppins, MdMuslins, MdSVWCs and MdCrustins), the linear α-helical peptides (MdCecropins) and the specific amino acid-rich peptides (MdDomesticins, MdDiptericins, MdEdins and MdAttacins). On this basis, we identified multiple genetic mechanisms that could have shaped the molecular and structural diversity of the M. domestica AMPs, including: (1) Gene duplication; (2) Exon duplication via shuffling; (3) Protein terminal variations; (4) Evolution of disulfide bridges via compensation. Our results not only enlarge the insect AMP family members, but also offer a basic platform for further studying the roles of such molecular diversity in contributing to the high efficiency of the housefly antimicrobial immune system.

Expression of antimicrobial peptide snakin-1 confers effective protection in rice against sheath blight pathogen, Rhizoctonia solani

Rice sheath blight disease caused by the necrotrophic fungus Rhizoctonia solani, is an economically detrimental fungal disease which significantly affects rice productivity worldwide. Potato snakin-1, a cysteine-rich basic antimicrobial peptide (AMP), is a member of the novel Snakin AMP family. In order to assess the ability of the snakin-1 peptide in protecting rice against the sheath blight disease, we have developed transgenic rice constitutively expressing the snakin-1 peptide. The antimicrobial activity of snakin-1 was evaluated against the sheath blight pathogen Rhizoctonia solani both in vitro and in planta. Crude protein from transgenic rice leaves showed in vitro antifungal activity against Rhizoctonia solani. Moreover, in planta bioassay results also confirmed the same, wherein snakin-1 expressing rice plants showed significantly enhanced protection against the sheath blight disease. This report demonstrates how a member of the Snakin family of antimicrobial peptides has been successfully used to generate sheath blight resistance in rice, without compromising on its agronomic characteristics and at no phenotypic cost.

Structure Elucidation and Functional Studies of a Novel β-hairpin Antimicrobial Peptide from the Marine Polychaeta Capitella teleta.

Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play a key role in host defense. Among the most active and stable under physiological conditions AMPs are the peptides of animal origin that adopt a β-hairpin conformation stabilized by disulfide bridges. In this study, a novel BRICHOS-domain related AMP from the marine polychaeta Capitella teleta, named capitellacin, was produced as the recombinant analogue and investigated. The mature capitellacin exhibits high homology with the known β-hairpin AMP family—tachyplesins and polyphemusins from the horseshoe crabs. The β-hairpin structure of the recombinant capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide exists as monomeric right-handed twisted β-hairpin and its structure does not reveal significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment and incorporates into lipid bilayer. Capitellacin exhibits a strong antimicrobial activity in vitro against a wide panel of bacteria including extensively drug-resistant strains. In contrast to other known β-hairpin AMPs, this peptide acts apparently via non-lytic mechanism at concentrations inhibiting bacterial growth. The molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation therefore other molecular targets may be assumed. The reduced cytotoxicity against human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make it an attractive candidate compound for an anti-infective drug design.

(Re)Defining the Proline-Rich Antimicrobial Peptide Family and the Identification of Putative New Members.

As we rapidly approach a post-antibiotic era in which multi-drug resistant bacteria are ever-pervasive, antimicrobial peptides (AMPs) represent a promising class of compounds to help address this global issue. AMPs are best-known for their membrane-disruptive mode of action leading to bacteria cell lysis and death. However, many AMPs are also known to be non-lytic and have intracellular modes of action. Proline-rich AMPs (PrAMPs) are one such class, that are generally membrane permeable and inhibit protein synthesis leading to a bactericidal outcome. PrAMPs are highly effective against Gram-negative bacteria and yet show very low toxicity against eukaryotic cells. Here, we review both the PrAMP family and the past and current definitions for this class of peptides. Computational analysis of known AMPs within the DRAMP database (http://dramp.cpu-bioinfor.org/) and assessment of their PrAMP-like properties have led us to develop a revised definition of the PrAMP class. As a result, we subsequently identified a number of unknown and unclassified peptides containing motifs of striking similarity to known PrAMP-based DnaK inhibitors and propose a series of new sequences for experimental evaluation and subsequent addition to the PrAMP family.

Identification and characterization of novel bi-functional cathelicidins from the black-spotted frog (Pelophylax nigromaculata) with both anti-infective and antioxidant activities

Cathelicidins are an important family of antimicrobial peptides (AMPs), which play pivotal roles in vertebrate immune responses against microbial infections. They are regarded as potential drug leads for the development of novel antimicrobial agents and three related drugs have been developed into clinical trials. Thus, it is meaningful to identify more cathelicidins from vertebrate species. Cathelicidins from ranid frogs possess special structural characteristics and activities, but to date only 12 ranid frog cathelicidins have been identified. In the present study, two novel cathelicidins (PN-CATH1 and 2) were identified from the black-spotted frog, Pelophylax nigromaculata. PN-CATHs possess low sequence similarity with the known cathelicidins. They exhibited moderate, but broad-spectrum and rapid antimicrobial activities against the tested bacteria. They kill bacteria by mainly inducing bacterial membrane disruption and possibly generating intracellular ROS formation. They also possess potent anti-biofilm and persister cell killing activity, indicating their potential in combating infections induced by biofilms-forming bacteria. Besides direct antimicrobial activity, they exhibited potent anti-inflammatory activity by effectively inhibiting the LPS-induced production of pro-inflammatory cytokines in mouse macrophages, which could be partly ascribed to their direct LPS-neutralizing ability. Furthermore, PN-CATHs demonstrated powerful in vitro free radical scavenging activities. Ultraviolet radiation significantly increased their in vivo gene expression in frog skin. Meanwhile, they possess weak cytotoxic activity and extremely low hemolytic activity. PN-CATHs represent the first discovery of cathelicidins family AMPs with both potent anti-infective and antioxidant activities. The discovery of PN-CATHs provides potential peptide leads for the development of novel anti-infective and antioxidant drugs.

Identification of New Ocellatin Antimicrobial Peptides by cDNA Precursor Cloning in the Frame of This Family of Intriguing Peptides.

Ocellatins are a family of antimicrobial peptides found exclusively in the Leptodactylus genus. To date, 10 species have been studied and more than 23 peptides described. Here we report the sequences of five new peptides from the skin of the frog Leptodactylus latrans (Anura: Leptodactylidae) determined by cDNA cloning of the complete prepro-peptide structures. The mature peptides were characterized with in silico tools and compared with those previously described. With 21 amino acid residues, this new set of peptides not previously described in the Leptodactylus genus share between 100 and 76.2% similarity to ocellatin antimicrobial peptides. These novel peptides are cationic and their three-dimensional (3D) structure holds the highly conserved residues G1, D4, K7, and K11 and a high theoretical amphipathic α-helix content. Furthermore, in silico analyses of these new peptides predicted antimicrobial activity. This study is framed in the context of previous work published about ocellatins, and therefore, provides a review of this intriguing family of peptides.