Antimicrobial Peptide Defenses in Amphibian Skin

全球两栖动物数量的下降是一个令人严重关切的问题。几十年前，雷切尔·卡森引起了全世界对遭受滴滴涕影响的野生鸟类的关注。她的书提出了一个“寂静的春天”的黯淡前景，没有鸟儿欢快的合唱。今天，我们面临着一个新的寂静春天的前景，因为世界上五颜六色、引人注目的两栖动物数量继续下降，甚至消失。全球两栖动物数量下降的原因尚不清楚。最近的研究表明，这种下降与一种或多种皮肤侵袭性病原体有关。在两栖动物使用的免疫防御机制中，有一种是在皮肤的特殊颗粒腺(也称为毒腺)中产生抗菌肽。当两栖动物受伤或感觉到危险时，这些天然抗生素包可以倒在皮肤上。尽管由两栖动物皮肤制造和分泌的抗菌肽被认为对环境病原体具有保护作用，但很少有直接证据证明这一点。这里提出的研究旨在检验这样一种假设，即皮肤中产生的抗菌肽是以足够的浓度合成、加工、储存和分泌，以发挥皮肤防御皮肤直接环境中存在的病原体的作用。该项目将解决有关这些免疫防御的一些具体问题，并提供对这一重要的先天免疫防御机制的更深入的了解。第一个目标是检测静息状态下模型两栖动物皮肤表面抗菌肽的相对丰度和分泌模式，并与刺激交感神经系统从而模拟捕食者自然攻击的药物诱导的最大分泌水平进行比较。这一分析将通过使用质谱仪(MS)来完成，在MS中，一种高度复杂的仪器根据其独特的分子量将单个多肽从多肽混合物中分离出来。这些研究将建立可重复的采样和分析方案，并为其他研究提供基线数据。其他实验旨在消耗一些青蛙的抗菌皮肤肽，并与其他含有全套皮肤肽的青蛙进行比较，以检验这些青蛙对皮肤病原体感染的敏感性。第二个目标是研究两栖动物模型在休息和释放多肽后皮肤中抗菌肽前体的合成。多肽的直接前体是从DNA转录而来的信使RNA。这些研究将检查稳态合成的水平，以及在分泌多肽后诱导新合成的动力学。它们将为研究环境因素如何影响抗菌肽的合成提供基线数据。第三个目标是研究寒冷温度、糖皮质激素(应激激素)和局部炎症反应对皮肤抗菌肽合成和分泌的影响。这些研究旨在确定环境“应激源”是否会抑制抗菌肽的自然产生，从而使两栖动物宿主更容易感染疾病。

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.