Antimicrobial Peptide Defenses in Amphibian Skin

全球两栖动物数量的减少引起了极大的关注。几十年前，雷切尔·卡森（Rachel Carson）引起了全世界对野生鸟类遭受滴滴涕影响的困境的关注。她的书提出了一个“寂静的春天”的严峻前景，没有歌鸟的乐观合唱。今天，我们正面临着一个新的寂静的春天的前景，因为世界上色彩缤纷、引人注目的两栖动物的数量继续下降，甚至消失。全球两栖动物数量减少的原因尚不清楚。最近的研究表明，一种或多种皮肤侵入性病原体与这种下降有关。两栖动物使用的免疫防御机制之一是在皮肤上的特殊颗粒腺（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，这些天然抗生素可以被排空到皮肤上。虽然由两栖动物皮肤产生和分泌的抗菌肽被认为在保护免受环境病原体的侵害方面发挥作用，但很少有直接证据证明这一点。本文提出的研究旨在验证以下假设：皮肤中产生的抗菌肽被合成、加工、储存和分泌到足够的浓度，在保护皮肤免受皮肤直接环境中病原体的侵害中发挥作用。该项目将解决有关这些免疫防御的一些具体问题，并对这一重要的先天免疫防御机制提供更深入的了解。第一个目标是检查静止状态下模型两栖动物皮肤表面抗菌肽分泌的相对丰度和模式，并与刺激交感神经系统的药物引起的最大分泌后的水平进行比较，从而模拟捕食者的自然攻击。这种分析将通过使用质谱（MS）来完成，其中一个高度复杂的仪器根据肽的独特分子量从肽混合物中分离出单个肽。这些研究将建立可重复的抽样和分析方案，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，并与含有全套皮肤肽的其他青蛙相比，检查这些青蛙对皮肤病原体感染的易感性。第二个目的是研究模型两栖动物在休息和释放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水平，以及肽分泌后诱导新合成的动力学。它们将为环境因素如何影响抗菌肽合成的研究提供基线数据。第三个目的是研究低温、糖皮质激素（应激激素）和局部炎症反应对皮肤中抗菌肽合成和分泌的影响。这些研究旨在确定环境“压力源”是否可能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.