Skin peptides of different life stages of Ewing's tree frog

Abstract

In frogs, an important mechanism of skin innate immunity against invading microbial pathogens is secretion of antimicrobial peptides from the specialized granular glands. Since these glands develop fully in skin dermis after completion of metamorphosis, they are small and immature in skin of larvae (tadpoles). Skin secretions vary among different life stages. Antimicrobial activity and peptide composition of natural mixture of skin peptides of three different life stages of New Zealand Ewing's Tree Frog (Litoria ewingii), tadpoles, metamorphs and adults were analyzed. The peptide mixtures were collected from skin secretions and analyzed for activity against the standard reference bacterium, Escherichia coli (ATCC 25922). Their peptide components were analyzed using liquid chromatography mass spectrometry (LC-MS). The peptide mixture from adults and metamorphs contained the species-specific antimicrobial peptide uperin 7.1 and inhibited the growth of E. coli (ATCC 25922). In contrast, the peptide mixture of tadpoles did not inhibit the growth of E. coli (ATCC 25922). This peptide mixture did not contain uperin 7.1 but had peptides whose molecular masses did not correspond to molecular masses of any known frog antimicrobial peptides.

Functional analysis for gut microbes of the brown tree frog (Polypedates megacephalus) in artificial hibernation

“…For example, RLS, an infectious disease caused by septicemia, was the main cause of frog mortality [38–40]. Pathogens that increase the risk of RLS included Pseudomonas aeruginosa and Staphylococcus epidermidis [39, 40, 58]. Although, the RLS-related pathogens in brown tree frogs are still uncharacterized.…”

Skin micro-organs from several frog species secrete a repertoire of powerful antimicrobials in culture

“…It is has been suggested that every species harbors a unique, specific collection of antimicrobial peptides, tuned to defend the organism against microorganisms that it is likely to encounter. 13 As shown in Table 1, we do not seem to find any specific pattern, which assigns specific activities to certain habitats. In fact what seems to be the case is the opposite.…”

“…It is this line of though that directed our approach not towards individual substances but rather to a whole repertoire of substances that act in synergism, with the mixture having up to a 10-fold greater antibiotic activity than the peptides separately. 13,22 The methodology presented here allows for in vitro production, analysis and characterization of the 'natural mixture' of compounds secreted by different anuran species in order to cope with the highly hostile environment in which they live. In this respect it should be pointed out that some of the activities reported here, as shown in Figures 5 and 6, were very potent against various pathogenic bacteria without having any toxic effects on somatic cells even at 10-fold higher concentrations.…”

“…The frogs were kept alive in a purpose-built amphibian facility at 25 ± 1 1C under a 12-h light/dark cycle and fed with fruit flies three times per week. 13 All procedures involving frogs adhered to resolution 008430 of the Ministry of Health for the use of animals in research and the guidelines of the ethical committee on animal research from the Andes University. All captures were performed under the collection license No.…”

“…The positive control wells received SMO culture media (serum-free 70% DMEM (Sigma-Aldrich) instead of CM and negative control wells received 2 mg ml À1 of ampicillin. 13 Each test was performed in duplicate for each experimental condition. Plates were incubated at 37 1C and the OD 595 was read at three different time points: 6, 8 and 24 h.…”