Antimicrobial peptide defenses of the Tarahumara frog, Rana tarahumarae

https://doi.org/10.1016/S0006-291X(02)02217-9Get rights and content

Abstract

Populations of the Tarahumara frog Rana tarahumarae have decreased markedly in recent years in the northern part of their range. Infection by the chytrid fungus Batrachochytrium dendrobatidis has been implicated in these declines. To determine whether antimicrobial peptides in the skin provide protection against this pathogen, norepinephrine-stimulated skin secretions were tested for their ability to inhibit growth of B. dendrobatidis in vitro. After concentration, crude mixtures of skin peptides inhibited the growth of the chytrid in a concentration-dependent manner. Proteomic analysis led to the identification and characterization of three peptides belonging to the brevinin-1 family of antimicrobial peptides and three belonging to the ranatuerin-2 family. The two most abundant peptides, ranatuerin-2TRa (GIMDSIKGAAKEIAGHLLDNLKCKITGC) and brevinin-1TRa (FLPVIAGIAANVLPKLFCKLTKRC), were active against B. dendrobatidis (MIC of 50 μM for ranatuerin-2TRa and 12.5 μM for brevinin-1TRa against zoospores). These data clearly show that antimicrobial peptides in the skin secretions of the Tarahumara frog are active against B. dendrobatidis and should provide some protection against infection. Therefore, the observed susceptibility of these frogs to this pathogen in the wild may be due to the effects of additional environmental factors that impair this innate defense mechanism, leading to the observed population declines.

Section snippets

Materials and methods

Animals. Rana tarahumarae were collected as an egg mass in Mexico by S. Hale. J. Rorabaugh of the US Fish and Wildlife Service raised the eggs to tadpoles and transferred some to Arizona State University. These animals were raised to maturity at Arizona State University and two frogs were transferred to the Rollins-Smith laboratory for peptide collection.

Collection of skin secretions. A single individual of R. tarahumarae (23.3 g) was injected bilaterally with 2 nmoles/gram body weight of

Purification of the peptides

The elution profile on a Vydac C-18 semipreparative column of the combined skin secretions and washings, after partial purification on Sep-Pak cartridges, is shown in Fig. 1. Analysis by electrospray mass spectrometry demonstrated that the fractions denoted by zone A contained a major component of mass 2898 (subsequently shown to be ranatuerin-2TRa). The fractions denoted by zone B contained a major component of mass 2614 (subsequently shown to be brevinin-1TRa). The fractions denoted by zone C

Discussion

The present study examined the activity of antimicrobial peptides from the Tarahumara frog against the chytrid fungus that has been associated with population declines in Australia, Central America, the Western US, and Europe [5], [6], [7], [27]. Our work illustrates the power of reversed-phase HPLC coupled with electrospray mass spectrometry for the separation and identification of peptide components in a complex mixture. The application of these techniques to analysis of the skin secretions

Acknowledgements

We thank Dr. Donald Babin, Creighton University, for amino acid analyses and Ms. Eva Lovas, Creighton University, for mass spectrometry measurements. Collection of R. tarahumarae eggs was supported by a grant to S. Hale from the US Fish and Wildlife Service. The current work was supported by a grant from Restoragen Inc., Lincoln, Nebraska (to J.M.C.), and by an NSF IRCEB Grant IBN- 9977063 (James Collins, P.I.) and NSF Grant IBN-0131184 (to L.R.-S.).

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