The ebb and flow of antimicrobial skin peptides defends northern leopard frogs (Rana pipiens) against chytridiomycosis

Many amphibian species are threatened with extinction by the emerging infectious disease, chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis. This unprecedented global crisis threatens to reduce the biodiversity of the entire amphibian class. The fungus invades the skin and impairs the uptake and retention of essential ions leading to cardiac arrest. Antimicrobial peptides (AMPs) secreted into the mucus of some amphibians are thought to be an important defense against chytridiomycosis. However, little is known about the quantities of AMPs secreted under natural conditions, whether they are sufficient to protect against this pathogen, and how they interact with commensal microbes. To understand how defensive peptides and skin microbes may interact, it is essential to know the precise quantities of AMPs present under natural conditions. Using matrix‐assisted laser desorption time‐of‐flight mass spectrometry and growth inhibition assays, we show that northern leopard frogs (Rana pipiens) at rest constitutively release low amounts of AMPs that inhibit B. dendrobatidis in vitro, and AMP defenses are elevated following a simulated predator attack. Using a synthetic peptide analogue of brevinin‐1Pb as an external control, we quantified the amounts of four previously described AMPs (brevinin 1Pa, brevinin‐1Pb, brevinin‐1Pd, and ranatuerin‐2P) at several time points after secretion. Once secreted onto the skin, the peptides are most active for 15 min, and small quantities persist for at least 2 h. Taken together, our data suggest that small amounts of AMPs are rapidly available and quite stable on the skin of R. pipiens. They are effective inhibitors of B. dendrobatidis at these low constitutive concentrations but degrade within 2 h, protecting the integrity of the skin and commensal bacteria.     

Change in food ingestion induces rapid shifts in the diversity of microbiota associated with cutaneous mucus of Atlantic salmon Salmo salar

To test the hypothesis that a switch in diet might cause changes in the abundance and composition of mucous‐dwelling microorganisms, a short‐term experiment was conducted with Atlantic salmon Salmo salar. Fish were fed on three different diets: pelleted S. salar feed, macroinvertebrates or pellets supplemented with an antibiotic. A fourth group of fish was deprived of food throughout the trial. Seven days after manipulating diets, significant differences were found in microbial density and community composition (quantified by different morphologically distinct colonies), particularly between fed and unfed animals. Moreover, food deprivation caused a rapid decrease in the number of epidermal mucous cells of the lateral skin, which may indicate a decrease in mucous secretion and explain differences in the diversity of mucous‐dwelling microbiota observed in the fish. This is the first report of an effect of feeding regime on the abundance of microbial communities associated with cutaneous mucus of fishes.     

A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines

Background

To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.

Results

Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.

Conclusions

The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.     

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

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

A Novel Lipopeptide from Skin Commensal Activates TLR2/CD36-p38 MAPK Signaling to Increase Antibacterial Defense against Bacterial Infection

Staphylococcus epidermidis (S.epidermidis) plays important protective roles by directly producing or by stimulating hosts to produce antimicrobial peptides (AMPs) against pathogenic infections. Although several AMPs from S.epidermidis have been identified, molecules that stimulate hosts to produce AMPs remain largly unknown. Here we demonstrate that a new lipopeptide (named LP01) purified from S.epidermidis culture media has a unique structure with heneicosanoic acid (21 carbons) binding to lysine¹¹ of a peptide chain. In vitro LP01 increased the expression of β-defensin 2(hBD2) and hBD3 in neonatal human epidermal keratinocytes(NHEK), leading to increased capacity of cell lysates to inhibit the growth of S.aureus. In vivo LP01 induced the expression of mouse β-defensin 4(mBD4) to decrease the survival of local S.aureus in skin and systemic S.aureus survival in liver. The induction of beta-defensins by LP01 was dependent on TLR2 as Tlr2-deficient mice had decreased mBD4. Furthermore, knockdown of CD36 decreased the expression of hBD2 and hBD3, and p38 MAPK inhibitor significantly inhibited the expression of hBDs induced by LP01.Taken together, these findings demonstrate that lipopeptide LP01 from normal commensal S.epidermidis increases antimicrobial peptide hBD2 and hBD3 expression via the activation of TLR2/CD36-p38 MAPK, thus enhancing antimicrobial defense against pathogenic infections.     

Highly selective end-tagged antimicrobial peptides derived from PRELP

Background

Antimicrobial peptides (AMPs) are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens.

Methodology and Principal Findings

Antibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP). The optimized peptides were antimicrobial against a range of Gram-positive S. aureus and Gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH₂ was effective against various “superbugs” including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection.

Conclusions/Significance

Hydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent activity against multiresistant bacteria and efficiency in ex vivo wound infection models. A precise “tuning” of toxicity and proteolytic stability may be achieved by changing tag-length and adding W- or F-amino acid tags.     

The Interaction of Helicobacter pylori with the Adherent Mucus Gel Layer Secreted by Polarized HT29-MTX-E12 Cells

Helicobacter pylori colonises the gastric mucosa of humans. The majority of organisms live in mucus. These organisms are an important reservoir for infection of the underlying epithelium. Cell culture models for H. pylori infection do not normally possess a mucus layer. The interaction of H. pylori with TFF1, a member of the trefoil factor family found in gastric mucin, is mediated by lipopolysaccharide. To test the hypothesis that the interaction of H. pylori with TFF1 promotes mucus colonization we characterised the interaction of H. pylori with a mucus secreting cell line, HT29-MTX-E12. An isogenic mutant of H. pylori with truncated core oligosaccharides was produced and binding to TFF1 and ability to colonise HT29-MTX-E12 cells determined. The adherent mucus layer of HT29-MTX-E12 cells contained the gastric mucin MUC5AC and trefoil factors, TFF1 and TFF3. H. pylori was found within the mucus layer in discrete clusters and in close association with TFF1. It also interacted with the membrane bound mucin MUC1 and replicated when co-cultured with the cells. An isogenic mutant of H. pylori with a truncated LPS core did not interact with TFF1, and colonization of HT29-MTX-E12 cells was reduced compared to the wild-type strain (p<0.05). Preincubation of cells with wild type LPS but not with truncated LPS resulted in reduced colonization by H. pylori. These results demonstrate that the interaction of TFF1 with H. pylori is important for colonization of gastric mucus and the core oligosaccharide of H. pylori LPS is critical for this interaction to occur. HT29-MTX-E12 cells are a useful system with which to study the interaction of bacteria with mucosal surfaces and the effect of such interactions on mediating colonization.     

The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH₂, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025–4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH₂ now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH₂ to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.     

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.     

Elicited cross-protection and specific antibodies in Mozambique tilapia (Oreochromis mossambicus) against two different immobilization serotypes of Cryptocaryon irritans isolated in Hawaii

The objective of this study was to determine whether immunization of Mozambique tilapia with different Cryptocaryon irritans i-antigen serotypes elicited cross-protection against challenge infection by both serotypes. Fish were directly exposed to live theronts of isolate W1 or isolate K1, that express different surface i-antigens. There was no significant difference in the number of trophonts infecting the fish between the two isolates, W1 and K1, following primary exposure. Serum from immunized fish exposed to live theronts showed higher immobilization titres and ELISA values against homologous isolates than to heterologous isolates after the primary exposure. However, mucus antibody did not immobilize theronts although the ELISA results clearly indicated that mucus antibodies recognizing C. irritans were generated. In a study with Western blot analyses, serum antibodies recognized only an antigen of the corresponding serotype and no proteins common to both serotypes were identified. Sequence analyses of 754 bases of rDNA nucleotide sequence including complete nuclear ribosomal ITS-1–5.8S rDNA–ITS-2 region were conducted and found to be identical for W1- and K1-isolates. These findings confirmed that both isolates were members of the species, C. irritans, and that rDNA analysis would not distinguish the two isolates. In conclusion, despite the fact that the immobilization assays and ELISA detected two serotypes in vitro, challenge assays provided evidence for only one type of C. irritans.     

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

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

Hilyses®, fermented Saccharomyces cerevisiae, enhances the growth performance and skin non-specific immune parameters in rainbow trout (Oncorhynchus mykiss)

Effects of Hilyses^®, fermented Saccharomyces cerevisiae (S. cerevisiae), on growth, body composition and skin mucus immune components in rainbow trout were quantified. Ninety rainbow trout (105 ± 5 g) were randomly assigned to 2 groups in triplicates and fed dietary Hilyses^® (5 g kg^?1) or control diet without Hilyses^® for 50 days. Results of this study demonstrated that growth performance increased significantly by the dietary yeast supplement; however body composition was not affected in treatment group. At the 45th and 50th day of feeding trial, results of mucus samples demonstrated that yeast supplementation in treatment group significantly promoted enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase compared to control group. Significant increases were also observed in hemagglutination and antibacterial activity against Yersinia ruckeri in fish fed treatment diet. The present study suggests that fermented S. cerevisiae may effectively promote the growth performance and skin non-specific immune parameters in rainbow trout.     

Antimicrobial properties and death-inducing mechanisms of saccharomycin,a biocide secreted by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.     

Stability of Microbiota Facilitated by Host Immune Regulation: Informing Probiotic Strategies to Manage Amphibian Disease

Microbial communities can augment host immune responses and probiotic therapies are under development to prevent or treat diseases of humans, crops, livestock, and wildlife including an emerging fungal disease of amphibians, chytridiomycosis. However, little is known about the stability of host-associated microbiota, or how the microbiota is structured by innate immune factors including antimicrobial peptides (AMPs) abundant in the skin secretions of many amphibians. Thus, conservation medicine including therapies targeting the skin will benefit from investigations of amphibian microbial ecology that provide a model for vertebrate host-symbiont interactions on mucosal surfaces. Here, we tested whether the cutaneous microbiota of Panamanian rocket frogs, Colostethus panamansis, was resistant to colonization or altered by treatment. Under semi-natural outdoor mesocosm conditions in Panama, we exposed frogs to one of three treatments including: (1) probiotic - the potentially beneficial bacterium Lysinibacillus fusiformis, (2) transplant – skin washes from the chytridiomycosis-resistant glass frog Espadarana prosoblepon, and (3) control – sterile water. Microbial assemblages were analyzed by a culture-independent T-RFLP analysis. We found that skin microbiota of C. panamansis was resistant to colonization and did not differ among treatments, but shifted through time in the mesocosms. We describe regulation of host AMPs that may function to maintain microbial community stability. Colonization resistance was metabolically costly and microbe-treated frogs lost 7–12% of body mass. The discovery of strong colonization resistance of skin microbiota suggests a well-regulated, rather than dynamic, host-symbiont relationship, and suggests that probiotic therapies aiming to enhance host immunity may require an approach that circumvents host mechanisms maintaining equilibrium in microbial communities.     

Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions

Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.     

Solution structure and activation mechanism of ubiquitin-like small archaeal modifier proteins

In archaea, two ubiquitin-like small archaeal modifier protein (SAMPs) were recently shown to be conjugated to proteins in vivo. SAMPs display homology to bacterial MoaD sulfur transfer proteins and eukaryotic ubiquitin-like proteins, and they share with them the conserved C-terminal glycine-glycine motif. Here, we report the solution structure of SAMP1 from Methanosarcina acetivorans and the activation of SAMPs by an archaeal protein with homology to eukaryotic E1 enzymes. Our results show that SAMP1 possesses a β-grasp fold and that its hydrophobic and electrostatic surface features are similar to those of MoaD. M. acetivorans SAMP1 exhibits an extensive flexible surface loop between helix-2 and the third strand of the β-sheet, which contributes to an elongated surface groove that is not observed in bacterial ubiquitin homologues and many other SAMPs. We provide in vitro biochemical evidence that SAMPs are activated in an ATP-dependent manner by an E1-like enzyme that we have termed E1-like SAMP activator (ELSA). We show that activation occurs by formation of a mixed anhydride (adenylate) at the SAMP C-terminus and is detectable by SDS-PAGE and electrospray ionization mass spectrometry.     

Cross reactivity of S. aureus to murine cytokine assays: A source of discrepancy

Staphylococcus aureus is one of the versatile Gram positive bacteria causing a range of diseases. Upon challenge, host immune cells recognize S. aureus and mount diverse immune responses including production of pro-inflammatory cytokines such as IL-1β and TNF-α. These cytokines are important mediators of inflammation which can be detected via various immunological methods such as enzyme linked immunosorbent assay (ELISA) and immunoblotting. In the current study, we found that a number of clinical isolates as well as laboratory strains of S. aureus exhibited cross reactivity with ELISA antibodies for murine IL-1β and TNF-α assays. This cross reactivity generates exaggerated false positive signals which can be a source of discrepancy for the understanding of real immune responses against S. aureus infection by host immune cells.