Opossum Cathelicidins Exhibit Antimicrobial Activity Against a Broad Spectrum of Pathogens Including West Nile Virus.

This study aimed to characterize cathelicidins from the gray short-tailed opossum in silico and experimentally validate their antimicrobial effects against various pathogenic bacteria and West Nile virus (WNV). Genome-wide in silico analysis against the current genome assembly of the gray short-tailed opossum yielded 56 classical antimicrobial peptides (AMPs) from eight different families, among which 19 cathelicidins, namely ModoCath1 - 19, were analyzed in silico to predict their antimicrobial domains and three of which, ModoCath1, -5, and -6, were further experimentally evaluated for their antimicrobial activity, and were found to exhibit a wide spectrum of antimicroial effects against a panel of gram-positive and gram-negative bacterial strains. In addition, these peptides displayed low-to-moderate cytotoxicity in mammalian cells as well as stability in serum and various salt and pH conditions. Circular dichroism analysis of the spectra resulting from interactions between ModoCaths and lipopolysaccharides (LPS) showed formation of a helical structure, while a dual-dye membrane disruption assay and scanning electron microscopy analysis revealed that ModoCaths exerted bactericidal effects by causing membrane damage. Furthermore, ModoCath5 displayed potent antiviral activity against WNV by inhibiting viral replication, suggesting that opossum cathelicidins may serve as potentially novel antimicrobial endogenous substances of mammalian origin, considering their large number. Moreover, analysis of publicly available RNA-seq data revealed the expression of eight ModoCaths from five different tissues, suggesting that gray short-tailed opossums may be an interesting source of cathelicidins with diverse characteristics.

Characterization of a Cathelicidin from the Colubrinae Snake, Sinonatrix annularis.

The cathelicidins represent an important family of host defense peptides (HDPs) found exclusively in vertebrates, which serve as a critical effector in host immune response against microbial infections. To date, a large number of cathelicidins has been identified from diverse vertebrates, such as mammals, birds, reptiles, amphibians and fishes. Sixteen cathelicidins have been identified from snakes in the Elapidae, Viperidae, and Biodae families. However, no cathelicidin has been discovered from a snake of the Colubrinae family. In the present study, we report the identification and characterization of a novel cathelicidin, SA-CATH, from the Colubrinae family snake, Sinonatrix annularis. The cDNA sequence encoding SA-CATH precursor is 735 bp in length, and the mature peptide (SA-CATH) is composed of 30 amino acid residues. Sequence alignment result indicated that SA-CATH precursor possesses relatively high sequence similarity with the cathelicidins from Elapidae and Viperidae family snakes. Similar as the cathelicidins from Elapidae, Viperidae, and Biodae family snakes, SA-CATH mainly assumes an amphipathic alpha-helical conformation, and possesses potent antimicrobial, biofilm inhibitory and anti-inflammatory activities. The results in the present study imply that cathelicidins serve as a kind of conserved effectors with similar structures in the immune systems of Colubrinae, Boidae, Elapidae and Viperidae family snakes. The identification of SA-CATH provides novel clues for the understanding of function and evolution of snake immune systems. The potent antimicrobial, biofilm inhibitory, anti-inflammatory, and slight cytotoxic activities of SA-CATH imply that it is a potential drug candidate in novel antimicrobial agent development.

Molecular Farming in Barley: Development of a Novel Production Platform to Produce Human Antimicrobial Peptide LL-37.

The peptide LL-37, a component of the human innate immune system, represents a promising drug candidate. In particular, the development of low-cost production platform technology is a critical bottleneck in its use in medicine. In the present study, a viable approach for the LL-37 production in transgenic barley is developed. First, comparative analyses of the effects of different fused peptide epitope tags applicable for accumulation and purification on LL-37 production yield are performed using transient expression in tobacco leaves. Following the selection of the most yielding fusion peptide strategies, eight different constructs for the expression of codon optimized chimeric LL-37 genes in transgenic barley plants are created. The expression of individual constructs is driven either by an endosperm-specific promoter of the barley B1 hordein gene or by the maize ubiquitin promoter. The transgenes are stably integrated into the barley genome and inherited in the subsequent generation. All transgenic lines show normal phenotypes and are fertile. LL-37 accumulated in the barley seeds up to 0.55 mg per 1 kg of grain. The fused epitope tags are cleaved off by the use of enterokinase. Furthermore, in planta produced LL-37 including the fused versions is biologically active.

Identification of the first cathelicidin gene from skin of Chinese giant salamanders Andrias davidianus with its potent antimicrobial activity.

Cathelicidins, as effector molecules, play important roles against infections and represent a crucial component of the innate immune system in vertebrates. They are widely studied in mammals, but little is known in amphibians. In the present study, we report the identification and characterization of a novel cathelicidin from Chinese giant salamander Andrias davidianus, which is the first study in Caudata amphibian. The cDNA sequence encodes a predicted 148-amino-acid polypeptide, which composed of a 20-residue signal peptide, a 94-residue conserved cathelin domain and a 34-residue mature peptide. From the multiple sequence alignments and phylogenetic analysis, AdCath shared conserved structure with other orthologs and clustered with other amphibian peptides. The tissue expression profiles revealed AdCath was highly expressed in skin. To study the function of AdCath gene, the AdCath precursor protein and mature peptide were recombinantly expressed and chemical synthesized respectively. The rAdCath protein could bind to LPS in a dose-dependent manner. When the concentrations of rAdCath protein and mature peptide were up to 22 µg/mL, they showed significantly cytotoxicity to human 293T cell lines. The rAdCath protein and synthetic peptide could exhibit antibacterial activities detected by the minimum inhibitory concentrations assay. From the SEM assay, the synthetic mature peptide could destroy the membrane of bacteria and cause loss of membrane integrity. Collectively, these findings characterized the first cathelicidin from A. davidianus, and highlighted its potential antimicrobial activities, indicating its important roles in the skin immune response against different bacteria.

Antimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins.

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24-40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1-37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant ß-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture.

In vitro activity of human and animal cathelicidins against livestock-associated methicillin-resistant Staphylococcus aureus.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic pathogen. An emerging problem in treating S. aureus infections is the increasing resistance against antibiotics. A possible way to overcome this issue is to boost the host immune system and one target are antimicrobial peptides (AMPs), especially cathelicidins. The aim of this study was to characterize the antimicrobial activity of cathelicidins from different animal species against LA-MRSA and to reveal whether major antimicrobial resistance mechanisms influence the bactericidal activity of these peptides. The MICs of 153 LA-MRSA field isolates for different cathelicidins (LL-37, mCRAMP, CAP18, BMAP-27 and BMAP-28) were analysed. The cathelicidin MICs of S. aureus RN4220 and isogenic transformants, that carried 14 functionally active antimicrobial resistance genes, were determined. These resistance genes have been identified in LA-MRSA and specify the resistance mechanisms active efflux, enzymatic inactivation and modification/protection/replacement of target sites. The data showed that mode MIC values for the cathelicidins did not differ among the LA-MRSA isolates of different animal origin. However, distinct differences were detected between the MIC values for the different cathelicidins. MIC values were lowest for bovine cathelicidins (BMAP-27 and BMAP-28) and highest for the human and mouse cathelicidins (LL-37 and mCRAMP). None of the tested antimicrobial resistance genes affected the antimicrobial activity of the cathelicidins. The findings obtained in this study support the hypothesis that cathelicidins might be a promising target to support the host defense against LA-MRSA, especially since the antimicrobial activity of these peptides is not affected by common staphylococcal antimicrobial resistance genes.

Rhodamine B-conjugated encrypted vipericidin nonapeptide is a potent toxin to zebrafish and associated with in vitro cytotoxicity.

BACKGROUND: Animal venoms contain a diverse array of proteins and enzymes that are toxic toward various physiological systems. However, there are also some practical medicinal uses for these toxins including use as anti-bacterial and anti-tumor agents. METHODS: In this study, we identified a nine-residue cryptic oligopeptide, KRFKKFFKK (EVP50) that is repeatedly encoded in tandem within vipericidin sequences. RESULTS: EVP50 displayed in vivo potent lethal toxicity to zebrafish larvae (LD50=6 µM) when the peptide's N-terminus was chemically conjugated to rhodamine B (RhoB). In vitro, RhoB-conjugated EVP50 (RhoB-EVP50) exhibited a concentration-dependent cytotoxic effect toward MCF-7 and MDA-MB-231 breast cancer cells. In MCF-7 cells, the RhoB-EVP50 nonapeptide accumulated inside the cells within minutes. In the cytoplasm, the RhoB-EVP50 induced extracellular calcium influx and intracellular calcium release. Membrane budding was also observed after incubation with micromolar concentrations of the fluorescent EVP50 conjugate. CONCLUSIONS: The conjugate's interference with calcium homeostasis, its intracellular accumulation and its induced membrane dysfunction (budding and vacuolization) seem to act in concert to disrupt the cell circuitry. Contrastively, unconjugated EVP50 peptide did not display neither toxic nor cytotoxic activities in our in vivo and in vitro models. GENERAL SIGNIFICANCE: The synergic mechanism of toxicity was restricted to the structurally modified encrypted vipericidin nonapeptide.

Antimicrobial peptide Cathelicidin-BF prevents intestinal barrier dysfunction in a mouse model of endotoxemia.

Intestinal barrier functions are altered during the development of sepsis. Cathelicidin antimicrobial peptides, such as LL-37 and mCRAMP, can protect animals against intestinal barrier dysfunction. Cathelicidin-BF (C-BF), a new cathelicidin peptide purified from the venom of the snake Bungarus fasciatus, has been shown to have both antimicrobial and anti-inflammatory properties. This study investigated whether C-BF pretreatment could protect the intestinal barrier against dysfunction in a mouse model of endotoxemia, induced by intraperitoneal injection of LPS (10mg/kg). Mice were treated with low or high dose C-BF before treatment with LPS, and samples were collected 5h after LPS treatment. C-BF reduced LPS induced intestinal histological damage and gut permeability to 4 KD Fluorescein-isothiocyanate-conjugated dextran. Pretreatment with C-BF prevented LPS induced intestinal tight junction disruption and epithelial cell apoptosis. Moreover, C-BF down regulated the expression and secretion of TNF-α, a process involving the NF-κB signaling pathway. C-BF also reduced LPS induced TNF-α expression through the NF-κB signaling pathway in mouse RAW 264.7 macrophages. These findings indicate that C-BF can prevent gut barrier dysfunction induced by LPS, suggesting that C-BF may be used to develop a prophylactic agent for intestinal injury in endotoxemia.

Helical cationic antimicrobial peptide length and its impact on membrane disruption.

Cationic antimicrobial peptides (CAMPs) are important elements of innate immunity in higher organisms, representing an ancient defense mechanism against pathogenic bacteria. These peptides exhibit broad-spectrum antimicrobial activities, utilizing mechanisms that involve targeting bacterial membranes. Recently, a 34-residue CAMP (NA-CATH) was identified in cDNA from the venom gland of the Chinese cobra (Naja atra). A semi-conserved 11-residue pattern observed in the NA-CATH sequence provided the basis for generating an 11-residue truncated peptide, ATRA-1A, and its corresponding D-peptide isomer. While the antimicrobial and biophysical properties of the ATRA-1A stereoisomers have been investigated, their modes of action remain unclear. More broadly, mechanistic differences that can arise when investigating minimal antimicrobial units within larger naturally occurring CAMPs have not been rigorously explored. Therefore, the studies reported here are focused on this question and the interactions of full-length NA-CATH and the truncated ATRA-1A isomers with bacterial membranes. The results of these studies indicate that in engineering the ATRA-1A isomers, the associated change in peptide length and charge dramatically impacts not only their antimicrobial effectiveness, but also the mechanism of action they employ relative to that of the full-length parent peptide NA-CATH. These insights are relevant to future efforts to develop shorter versions of larger naturally occurring CAMPs for potential therapeutic applications.

Expression and one-step purification of the antimicrobial peptide cathelicidin-BF using the intein system in Bacillus subtilis.

The intein expression system has been widely applied in Escherichia coli to express various proteins and peptides. However, the removal of endotoxin from the recombinant proteins expressed in E. coli is very difficult and therefore complicates the purification process. In this study, we constructed an intein-based expression vector for an antimicrobial peptide (cathelicidin from Bungarus fasciatus) and expressed the intein fusion peptide in a Bacillus subtilis expression system. The fusion peptide was secreted into the culture medium, identified by Western blot and purified by affinity chromatography and intein self-cleavage in just one step. Approximately, 0.5 mg peptide was obtained from 1 litre of culture medium. The purified peptide showed antimicrobial activity. Our results indicate that the intein expression system may be a safe and efficient method to produce soluble peptides and proteins in B. subtilis.

Effect of antimicrobial peptides derived from human cathelicidin LL-37 on Entamoeba histolytica trophozoites.

The human cathelicidin hCAP18/LL-37 is an antimicrobial protein consisting of a conserved N-terminal prosequence called the cathelin-like domain and a C-terminal peptide called LL-37. This peptide contains 37 amino acid residues, and several truncated variants obtained from natural sources or by chemical synthesis differ in their capability to damage Gram positive and Gram negative bacteria as well as Candida albicans. KR-12 is the shortest peptide (12 amino acids) of LL-37 that has conserved antibacterial activity. In addition to LL-37, other active cathelicidin-derived peptides have been reported; for instance, the peptides KR-20, a 20-aa derivative of LL-37, and KS-30, a 30-aa derivative of LL-37, have been found in human sweat. Both peptides exhibit an overall increased antibacterial and antifungal activity when compared with LL-37. We investigated the effect of LL-37 and three peptides derived from this antimicrobial molecule, KR-12, KR-20 and KS-30, on the integrity of Entamoeba histolytica trophozoites. The four peptides showed effects on E. histolytica integrity and viability in the concentration range of 10-50 µM. The peptides KR-12, KR-20, KS-30 and LL-37 differed in their capability to damage the parasite integrity, with KR-20 being the most effective and with KR-12 and LL-37 being less active. These results demonstrate the ability of antimicrobial peptides derived from human cathelicidin to damage Entamoeba trophozoites. Moreover, it was shown that the integrity of the peptides is altered in the presence of an ameba soluble fraction with cysteine protease activity.

A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein.

LL-37 is the only cathelicidin-derived antimicrobial peptide found in humans and it has a multifunctional role in host defense. The peptide has been shown to possess immunomodulatory functions in addition to antimicrobial activity. To provide sufficient material for biological and structural characterization of this important peptide, various systems were developed to produce recombinant LL-37 in Escherichia coli. In one previous approach, LL-37 coding sequence was cloned into vector pET-32a, allowing the peptide to be expressed as a thioredoxin fusion. The fusion protein contains two thrombin cleavage sites: a vector-encoded one that is 30-residue upstream of the insert and an engineered one that is immediately adjacent to LL-37. Cleavage at these two sites shall generate three fragments, one of which is the target peptide. However, when the fusion protein was treated with thrombin, cleavage only occurred at the remote upstream site. A plausible explanation is that the thrombin site adjacent to LL-37 is less accessible due to the peptide's aggregation tendency and cleavage at the remote site generates a fragment, which forms a large aggregate that buries the intended site. In this study, I deleted the vector-encoded thrombin site and S tag in pET-32a, and then inserted the coding sequence for LL-37 plus a thrombin site into the modified vector. Although removing the S tag did not change the oligomeric state of the fusion protein, deletion of the vector-encoded thrombin site allowed the fusion to be cleaved at the engineered site to release LL-37. The released peptide was separated from the carrier and cleavage enzyme by size-exclusion chromatography. This new approach enables a quick production of high quality active LL-37 with a decent amount.

Recombinant production of antimicrobial peptides in Escherichia coli: a review.

Antimicrobial peptides are of great interest due to their potential application as novel antibiotics. Large quantities of highly purified peptides are required to meet the needs of basic research and clinical trials. Compared with isolation from natural sources and chemical synthesis, recombinant approach offers the most cost-effective means for large-scale peptide manufacture. Among the systems available for heterologous protein production, Escherichia coli has been the most widely used host. Antimicrobial peptides produced in E. coli are often expressed as fusion proteins, a strategy necessary to mask these peptides' lethal effect towards the host and protect them from proteolytic degradation. The present article reviews commonly used fusion partners (e.g., solubility-enhancing, aggregation-promoting and self-cleavable carriers, etc.), cleavage methods and optimization options for antimicrobial peptides production in E. coli. In addition, the various approaches developed to generate recombinant human antimicrobial peptide LL-37, which offer excellent examples demonstrating effective production strategies, were briefly discussed.

Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli.

Cationic antimicrobial host defense peptides (HDPs) combat infection by directly killing a wide variety of microbes, and/or modulating host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites, but there are substantial roadblocks to their therapeutic application. High manufacturing costs associated with amino acid precursors have limited the delivery of inexpensive therapeutics through industrial-scale chemical synthesis. Conversely, the production of peptides in bacteria by recombinant DNA technology has been impeded by the antimicrobial activity of these peptides and their susceptibility to proteolytic degradation, while subsequent purification of recombinant peptides often requires multiple steps and has not been cost-effective. Here we have developed methodologies appropriate for large-scale industrial production of HDPs; in particular, we describe (i) a method, using fusions to SUMO, for producing high yields of intact recombinant HDPs in bacteria without significant toxicity and (ii) a simplified 2-step purification method appropriate for industrial use. We have used this method to produce seven HDPs to date (IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6). Using this technology, pilot-scale fermentation (10L) was performed to produce large quantities of biologically active cationic peptides. Together, these data indicate that this new method represents a cost-effective means to enable commercial enterprises to produce HDPs in large-scale under Good Laboratory Manufacturing Practice (GMP) conditions for therapeutic application in humans.

Identification, characterization and expression of cathelicidin in the pouch young of tammar wallaby (Macropus eugenii).

Antimicrobial peptides, such as cathelicidin, are an evolutionarily old defense system. However they have more complex actions than just simply their antimicrobial effects, including immunoregulation and interaction with the adaptive immune system. In this study we have characterized several novel cathelicidin-like peptides from the tammar wallaby (Macropus eugenii). The tammar cathelicidin-like (MaeuCath) mRNA were isolated based on the conservation of the cathelin-like amino terminus. Mature MaeuCath peptides were positively charged with hydrophobic carboxyl tails, features that are fundamental for antimicrobial function. MaeuCath1 was induced in tammar leukocytes in response to pathogen-associated molecular patterns from both gram positive and negative bacteria. In addition, we also examined the expression of MaeuCath1 in the primary and secondary lymphoid organs of the tammar neonate throughout early pouch life. The results from this study demonstrate the importance that MaeuCath1 may play in innate defense of the marsupial young, especially in the mucosal organs. Such expression of antimicrobial peptides may form part of the immune strategies of marsupials for neonatal survival during their post-partum development.

Antimicrobial fragments of the pro-region of cathelicidins and other immune peptides.

In addition to the numerous cathelicidin peptides that are associated with the antimicrobial activity exhibited by a crude extract from ovine blood, a further three peptides with antimicrobial activity have been identified. These were part of the precursor cathelin domain of cathelicidins, a large fragment of platelet factor 4 and a small peptide similar to signal peptide of the T-cell glycoprotein CD4 precursor. Fragments of proteins that are involved in protecting the host from infection may have a secondary purpose as antimicrobial agents once they have carried out their primary purpose and are cleaved the main protein.