Antimicrobial Peptide Defenses in Amphibian Skin

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Global declines of amphibian populations are a source of great concern. Several decades ago, Rachel Carson drew the attention of the world to the plight of wild birds suffering from the effects of DDT. Her book raised the grim prospect of a "silent spring" without the optimistic choruses of song birds. Today, we are faced with the prospect of a new silent spring as the world's populations of colorful and remarkable amphibians continue to decline and even disappear. The causes of global amphibian declines are unknown. Recent studies have implicated one or more skin-invasive pathogens in the declines. Among the immune defense mechanisms used by amphibians is the production of antimicrobial peptides in specialized granular glands (also called poison glands) in the skin. These packets of natural antibiotics can be emptied onto the skin when the amphibian is injured or when danger is sensed. Although the antimicrobial peptides made and secreted by amphibian skin are thought to play a role in protection from environmental pathogens, there is very little direct evidence to demonstrate this. The studies proposed here are designed to test the hypothesis that antimicrobial peptides produced in the skin are synthesized, processed, stored, and secreted in sufficient concentrations to play a role in defense of the skin from pathogens present in the immediate environment of the skin. The project will address a number of specific questions about these immune defenses and provide greater insight into this important innate immune defense mechanism. The first objective is to examine the relative abundance and pattern of secretion of antimicrobial peptides on the skin surface of model amphibians at rest in comparison with the levels present following maximal secretion induced by agents that stimulate the sympathetic nervous system and thus mimic a natural attack by a predator. This analysis will be accomplished by using mass spectrometry (MS) in which a highly sophisticated instrument separates individual peptides from a mixture of peptides based on their unique molecular weights. These studies will establish reproducible sampling and analysis protocols and provide baseline data for additional studies. Other experiments are designed to deplete antimicrobial skin peptides in some frogs and examine the susceptibility of these frogs to infection by skin pathogens in comparison with other frogs that contain the full set of skin peptides.The second objective is to study the synthesis of precursors of antimicrobial peptides in the skin of model amphibians at rest and following discharge of peptides. The immediate precursors of peptides are messenger RNAs that have been transcribed from DNA. These studies will examine the levels of steady state synthesis, and kinetics of induction of new synthesis following secretion of peptides. They will provide baseline data for studies of how environmental factors may affect the synthesis of antimicrobial peptides.The third objective is to examine the effects of cold temperature, glucocorticoid hormones (stress hormones), and local inflammatory responses on synthesis and secretion of antimicrobial peptides in the skin. These studies are designed to determine whether environmental "stressors" may inhibit the natural production of antimicrobial peptides and thus render the amphibian host more susceptible to disease.