Antimicrobial Peptide defenses in amphibian skin

One of the most urgent problems in conservation biology todayis the continuing loss of amphibian populations on a globalscale. Recent amphibian population declines in Australia, CentralAmerica, the western United States, Europe, and Africa havebeen linked to a pathogenic chytrid fungus, Batrachochytriumdendrobatidis, which infects the skin. The skin of amphibiansis critical for fluid balance, respiration, and transport ofessential ions; and the immune defense of the skin must be integratedwith these physiological responses. One of the natural defensesof the skin is production of antimicrobial peptides in granularglands. Discharge of the granular glands is initiated by stimulationof sympathetic nerves. To determine whether antimicrobial skinpeptides play a role in protection from invasive pathogens,purified antimicrobial peptides and natural peptide mixturesrecovered from the skin secretions of a number of species havebeen assayed for growth inhibition of the chytrid fungus. Thegeneral findings are that most species tested have one or moreantimicrobial peptides with potent activity against the chytridfungus, and natural mixtures of peptides are also effectiveinhibitors of chytrid growth. This supports the hypothesis thatantimicrobial peptides produced in the skin are an importantdefense against skin pathogens and may affect survival of populations.We also report on initial studies of peptide depletion usingnorepinephrine and the kinetics of peptide recovery followinginduction. Approximately 80 nmoles/g of norepinephrine is requiredto deplete peptides, and peptide stores are not fully recoveredat three weeks following this treatment. Because many specieshave defensive peptides and yet suffer chytrid-associated populationdeclines, it is likely that other factors (temperature, conditionsof hydration, "stress," or pesticides) may alter normal defensesand allow for uncontrolled infection.     

Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses

Innate immune mechanisms of defense are especially important to ectothermic vertebrates in which adaptive immune responses may be slow to develop. One innate defense in amphibian skin is the release of abundant quantities of antimicrobial peptides. Chytridiomycosis is an emerging infectious disease of amphibians caused by the skin fungus, Batrachochytrium dendrobatidis . Susceptibility to chytridiomycosis varies among species, and mechanisms of disease resistance are not well understood. Previously, we have shown that Australian and Panamanian amphibian species that possess skin peptides that effectively inhibit the growth of B. dendrobatidis in vitro tend to survive better in the wild or are predicted to survive the first encounter with this lethal pathogen. For most species, it has been difficult to experimentally infect individuals with B. dendrobatidis and directly evaluate both survival and antimicrobial peptide defenses. Here, we demonstrate differences in susceptibility to chytridiomycosis among four Australian species ( Litoria caerulea, Litoria chloris, Mixophyes fasciolatus and Limnodynastes tasmaniensis ) after experimental infection with B. dendrobatidis , and show that the survival rate increases with the in vitro effectiveness of the skin peptides. We also observed that circulating granulocyte, but not lymphocyte, counts differed between infected and uninfected Lit. chloris . This suggests that innate granulocyte defenses may be activated by pathogen exposure. Taken together, our data suggest that multiple innate defense mechanisms are involved in resistance to chytridiomycosis, and the efficacy of these defenses varies by amphibian species.     

Antimicrobial peptide defenses in the salamander, Ambystoma tigrinum, against emerging amphibian pathogens

Skin peptides were collected from living Ambystoma tigrinum larvae and adults and tested against two emerging pathogens, Batrachochytrium dendrobatidis and the Ambystoma tigrinum virus (ATV), as well as bacteria isolated from A. tigrinum. Natural mixtures of skin peptides were found to inhibit growth of B. dendrobatidis, Staphylococcus aureus, and Klebsiella sp., but activity against ATV was unpredictable. Skin peptides collected from salamanders held at three environmentally relevant temperatures differed in activity against B. dendrobatidis. Activity of the A. tigrinum skin peptides was found to be strongly influenced by pH.     

Population trends associated with skin peptide defenses against chytridiomycosis in Australian frogs

Many species of amphibians in the wet tropics of Australia have experienced population declines linked with the emergence of a skin-invasive chytrid fungus, Batrachochytrium dendrobatidis. An innate defense, antimicrobial peptides produced by granular glands in the skin, may protect some species from disease. Here we present evidence that supports this hypothesis. We tested ten synthesized peptides produced by Australian species, and natural peptide mixtures from five Queensland rainforest species. Natural mixtures and most peptides tested in isolation inhibited growth of B. dendrobatidis in vitro. The three most active peptides (caerin 1.9, maculatin 1.1, and caerin 1.1) were found in the secretions of non-declining species (Litoria chloris, L. caerulea, and L. genimaculata). Although the possession of a potent isolated antimicrobial peptide does not guarantee protection from infection, non-declining species (L. lesueuri and L. genimaculata) inhabiting the rainforest of Queensland possess mixtures of peptides that may be more protective than those of the species occurring in the same habitat that have recently experienced population declines associated with chytridiomycosis (L. nannotis, L. rheocola, and Nyctimystes dayi). This study demonstrates that in vitro effectiveness of skin peptides correlates with the degree of decline in the face of an emerging pathogen. Further research is needed to assess whether this non-specific immune defense may be useful in predicting disease susceptibility in other species.     

A novel antimicrobial peptide from amphibian skin secretions of Odorrana grahami

A novel antimicrobial peptide named odorranain-NR was identified from skin secretions of the diskless odorous frog, Odorrana grahami. It is composed of 23 amino acids with an amino acid sequence of GLLSGILGAGKHIVCGLTGCAKA. Odorranain-NR was classified into a novel family of antimicrobial peptide although it shared similarity with amphibian antimicrobial peptide family of nigrocin. Odorranain-NR has an unusual intramolecular disulfide-bridged hexapeptide segment that is different from the intramolecular disulfide-bridged heptapeptide segment at the C-terminal end of nigrocins. Furthermore, the -AKA fragment at the C-terminal of odorranain-NR is also different from nigrocins. Three different cDNAs encoding two odorranain-NR precursors and only one mature odorranain-NR was cloned from the cDNA library of the skin of O. grahami. This peptide showed antimicrobial activities against tested microorganisms except Escherichia coli (ATCC25922). Its antimicrobial mechanisms were investigated by transmission electron microcopy. Odorranain-NR exerted its antimicrobial functions by various means depending on different microorganisms.     

Identification of susceptibility loci for skin disease in a murine psoriasis model

Psoriasis is a frequently occurring inflammatory skin disease characterized by thickened erythematous skin that is covered with silvery scales. It is a complex genetic disease with both heritable and environmental factors contributing to onset and severity. The CD18 hypomorphic PL/J mouse reveals reduced expression of the common chain of beta(2) integrins (CD11/CD18) and spontaneously develops a skin disease that closely resembles human psoriasis. In contrast, CD18 hypomorphic C57BL/6J mice do not demonstrate this phenotype. In this study, we have performed a genome-wide scan to identify loci involved in psoriasiform dermatitis under the condition of low CD18 expression. Backcross analysis of a segregating cross between susceptible CD18 hypomorphic PL/J mice and the resistant CD18 hypomorphic C57BL/6J strain was performed. A genome-wide linkage analysis of 94 phenotypically extreme mice of the backcross was undertaken. Thereafter, a complementary analysis of the regions of interest from the genome-wide screen was done using higher marker density and further mice. We found two loci on chromosome 10 that were significantly linked to the disease and interacted in an additive fashion in its development. In addition, a locus on chromosome 6 that promoted earlier onset of the disease was identified in the most severely affected mice. For the first time, we have identified genetic regions associated with psoriasis in a mouse model resembling human psoriasis. The identification of gene regions associated with psoriasis in this mouse model might contribute to the understanding of genetic causes of psoriasis in patients and pathological mechanisms involved in development of disease.     

Predicted beta-turns in peptide and glycopeptide anti-freezes

A myosin light chain kinase has been obtained in a partially purified form from human blood platelets and bovine brain. The kinase from both sources required Ca²⁺ and the modulator protein for its activity. The subunit molecular weight is approximately 105,000 daltons. These kinases are therefore similar to the smooth muscle kinase (Dabrowska, R., Aromatorio, D., Sherry, J. M. F., and Hartshorne, D. J. (1977) Biochem. Biophys. Res. Commun. 78, 1263–1272). It is suggested that the role of the myosin light chain kinase is similar in both muscle and non-muscle cells and serves to activate the contractile apparatus, via the phosphorylation of myosin, in response to an increase in the intracellular free Ca²⁺ concentration.     

Human skin pigmentation, migration and disease susceptibility

Human skin pigmentation evolved as a compromise between the conflicting physiological demands of protection against the deleterious effects of ultraviolet radiation (UVR) and photosynthesis of UVB-dependent vitamin D(3). Living under high UVR near the equator, ancestral Homo sapiens had skin rich in protective eumelanin. Dispersals outside of the tropics were associated with positive selection for depigmentation to maximize cutaneous biosynthesis of pre-vitamin D(3) under low and highly seasonal UVB conditions. In recent centuries, migrations and high-speed transportation have brought many people into UVR regimes different from those experienced by their ancestors and, accordingly, exposed them to new disease risks. These have been increased by urbanization and changes in diet and lifestyle. Three examples-nutritional rickets, multiple sclerosis (MS) and cutaneous malignant melanoma (CMM)-are chosen to illustrate the serious health effects of mismatches between skin pigmentation and UVR. The aetiology of MS in particular provides insight into complex and contingent interactions of genetic and environmental factors necessary to trigger lethal disease states. Low UVB levels and vitamin D deficiencies produced by changes in location and lifestyle pose some of the most serious disease risks of the twenty-first century.     

Chemotaxis and chemical defenses in seaweed susceptibility to herbivory

Recent studies have show that small marine herbivores with limited mobility (mesograzers) often feed on macroalgae chemically defended against fishes or sea-urchins. In order to verify the involved mechanisms of chemotaxis or chemical defense into this process in Brazilian littoral, two species of brown alga Dictyota menstrualis and Dictyota mertensii were studied against the limited mobility herbivores, the amphipod Parhyale hawaiensis and the crab Pachygrapsus transversus. These two species were studied in order to verify the action of their crude extracts in the defense and chemotaxis processes related to limited mobility of these herbivores. Feeding preference assays revealed that P. hawaiensis do not eaten these Dictyota species. P. transversus do not eaten D. menstrualis either, but consumed large amounts of D. mertensii. Chemical deterrent assays showed that extracts of these species act as feeding deterrent to both species of herbivores. In addition, chemotaxis assays demonstrated that both herbivores are significantly negative chemotactic probably due to the presence of complementary metabolites into artificial foods. Considering that both Dictyota species exhibit active extracts against these small herbivores, we suppose that the non-occurrence of these herbivore species in close relationship with the seaweeds D. menstrualis and D. mertensii may explain the defense action of both extracts related to these mesograzers.     

Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin

A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.     

Model of Alzheimer's disease amyloid-beta peptide based on a RNA binding protein

Although Alzheimer's Abeta peptide has been shown to form beta-sheet structure, a high-resolution molecular structure is still unavailable to date. A search for a sequence neighbor using Abeta(10-42) as the query in the Protein Data-Bank (PDB) revealed that an RNA binding protein, AF-Sm1 from Archaeoglobus fulgidus (PDB entry: 1i4k chain Z), shared 36% identical residues. Using AF-Sm1 as a template, we built a molecular model of Abeta(10-42) by applying comparative modeling methods. The model of Abeta(10-42) contains an antiparallel beta-sheet formed by residues 16-23 and 32-41. Hydrophobic surface constituted by residues 17-20 (LVFF) separates distinctly charged regions. Residues that interact with RNA in the AF-Sm1 crystal structure were found to be conserved in Abeta. Using a native gel we demonstrate for the first time that RNA can interact with Abeta and selectively retard the formation of fibrils or higher-order oligomers. We hypothesize that in a similar fashion to AF-Sm1, RNA interacts with Abeta in the beta-hairpin (beta-turn-beta) structure and prevents fibril formation.     

Quantitative measurement of Batrachochytrium dendrobatidis in amphibian skin

The ability to quantify infections provides a tool with which to perform comparative pathological research. The need exists for a simplistic standard method to compare infection levels of Batrachochytrium dendrobatidis, a major cause of global amphibian declines. Through examination of skin sloughs of the Cape river frog Afrana fuscigula, we present an accessible method that not only provides quantitative measurements of B. dendrobatidis, but also provides information that increases the confidence of detection through histological surveys. The method relies on the availability of live animals that are actively shedding skin. By employing a direct microscopic count of sporangia, it is possible to express infection in terms of density. Micro-spatial infection in the skin of A. fuscigula is characterised by significant differences in sporangium density among the different components of the foot, and by similar differences in site infection frequency. Notably, toe tips and tubercles contain higher infection densities and are more often infected than webbing or the base of the foot. This pattern of infection might facilitate disease transmission due to the increased exposure of these components to abrasion. Density data can be used with the Poisson frequency function to approximate binomial probabilities of detecting B. dendrobatidis through histology. The probability matrix produced for A. fuscigula indicated that foot-site selection for histology markedly influenced the number of sections required to detect B. dendrobatidis at a specific level of probability. Thus, examination of a test sample of skin tissue with direct-count quantification can help in planning the sampling of tissues for histological surveys.     

Hallmarks of atopic skin mimicked in vitro by means of a skin disease model based on FLG knock-down

Loss-of-function mutations in the filaggrin gene (FLG) are a strong predisposing factor for atopic dermatitis, although their relevance to the disease pathomechanism needs further elucidation. The generation of an in vitro model of atopic skin would not only permit further evaluation of the underlying pathogenetic mechanisms and the testing of new treatment options, but would also allow toxicological studies to be performed in a simple, rapid and inexpensive manner. In this study, we have knocked down FLG expression in human keratinocytes and created three-dimensional skin models, which we used to investigate the impact of FLG on epidermal maturation and on skin absorption and its response to irritation. Histopathological evaluation of the skin models showed impaired epidermal differentiation in the FLG knock-down model. In addition, skin irritation induced by an application of sodium dodecyl sulphate resulted in significantly higher lactate dehydrogenase leakage, and interleukin (IL)-6 and IL-8 levels, than in the control model. To assess the effect of filaggrin deficiency on skin absorption of topically applied agents, we quantified the percutaneous absorption of lipophilic and hydrophilic model drugs, finding clinical relevance only for lipophilic drugs. This study clearly demonstrates that important clinical characteristics of atopic skin can be mimicked by using in vitro skin models. The FLG knock-down construct is the first step toward an in vitro model that allows clinical and toxicological studies of atopic-like skin.     

Seasonal patterns of activity and community structure in an amphibian assemblage at a pond network with variable hydrology

We studied community structure and seasonal activity patterns in a system of four ponds with seasonally-variable hydrology at a Mediterranean area in central Italy. We used a set of field methods to assess species presence and relative frequency of observation. The network of ponds was inhabited by six species of amphibians, two salamanders and four frogs. The breeding phenology of the six species did not vary remarkably among ponds, but there were significant differences among species in use of ponds. Factorial analysis of pond similarity drawn from percentage composition of the amphibian fauna, revealed that each of the four ponds was treatable as independent units, with no influence of relative inter-pond distance. PCA analysis allowed us to spatially arrange the amphibian species into three main groups: two were monospecific groups (i.e., Triturus vulgaris and Bufo bufo) and the third consisted of those species that selected not only the largest-deepest ponds, but also the ephemeral ones (i.e., Triturus carnifex, Hyla intermedia, the green frogs and Rana dalmatina). Our results suggest that the inter-pond differences in riparian vegetation, water depth, aquatic vegetation structure/abundance, and soil composition may produce differences among pond ecological characteristics (i.e., water turbidity and temperature, shelter availability, abundance of oviposition micro-sites), which may in turn influence different patterns of use by amphibians. To our knowledge, this is the first study emphasizing the potential role of heterochrony in the maintenance of a high species richness in Mediterranean amphibian communities. Preservation of freshwater vertebrate biodiversity requires management and protection not only of the main ponds and water bodies but also the temporary and ephemeral shallow ponds.     

Aldosterone upregulates purinergic responses in larval amphibian skin epithelium

Bullfrog tadpoles respond to apical application of 100 microM amiloride, acetylcholine (ACh) or ATP with a sharp transient inward (apical to basolateral) cation current. In adult skin, amiloride blockable transepithelial Na+ transport is upregulated by the hormone aldosterone. Tadpoles were treated in vivo with aldosterone and changes in short circuit current (Isc) in response to apical application of ATP were determined. Bullfrog tadpoles were exposed to aldosterone (10(-6) M) for periods ranging from 3 h to 60 h. Skins from 60-h aldosterone-treated animals showed a two- to three-fold increase in apical ATP-activated short circuit current when compared to animals treated with vehicle alone. Sodium replacement with a large, nonpermeable cation resulted in no measurable increase in Isc after exposure to ligand, consistent with ATP activation of an inward cation current and not chloride efflux. Activation/desensitization time courses and treatment with blockers revealed no measurable differences between aldosterone-treated and non-treated skins. Activation by amiloride and ACh gave essentially identical results. Studies with RT/PCR showed significant increases over controls of levels of mRNA associated with P2X channels. Given these data, our working hypothesis is that all three ligands activate the same process that exhibits both purinergic and cholinergic characteristics. These data are consistent with aldosterone upregulation of ATP gated channels expressed in the apical membrane of larval frog skin.     

Viral susceptibility of skin fibroblasts from patients with Huntington disease.

Cultured skin fibroblasts from patients with Huntington disease (HD) and age-matched controls were tested for susceptibility to vesicular stomatitis virus (VSV) and transformation by Kirsten mouse sarcoma virus (KiMSV). The HD and control cells could not be distinguished on the basis of viral replication, plaque morphology, virus yield, or susceptibility to transformation by KiMSV. These findings suggest that the HD gene product, if expressed within peripheral tissue, does not selectively alter or interfere with viral replication.