OSIB: African lungfish CtxA, a toxin for skin defense during estivation

机体对极端环境变化的适应往往涉及粘膜屏障组织的剧烈变化。非洲肺鱼可以通过一种叫做“睡眠”的生理过程在长时间的干旱期生存下来。羽化包括皮肤的剧烈重塑和表皮层的脱落以形成茧。我们小组之前的工作表明，肺鱼茧是一个含有许多免疫细胞的活组织，这些免疫细胞可以捕获细菌，在脆弱的休眠状态下保护肺鱼身体免受病原体的入侵。因此，茧的形成涉及到自我造成的粘膜炎症，但茧形成的分子机制尚不清楚。该提案研究了一种名为Protop-CtxA的新分子，这是一种编码在肺鱼基因组中的毒素。这种毒素似乎是通过细菌或病毒的水平基因转移获得的。Protop-CtxA的表达在肺鱼皮肤和茧中高度上调。我们假设这种分子足以在肺鱼孵化过程中破坏皮肤的完整性，并以这种方式形成茧。这项工作的目的是研究在自由游动和生长的肺鱼中哪些细胞产生Protop-CtxA，并确定Protop-CtxA的炎症、抗菌和杀虫功能。这项工作将提高我们对脊椎动物极端生理适应的认识，并且由于这种毒素的生物活性，可能会导致许多制药和农业应用。更广泛的影响包括外展活动，如阿尔伯克基自然历史博物馆的西班牙语和英语肺鱼展览，以及昆姆儿童时间的广播播客。非洲肺鱼（Protopterus sp.）既生活在水中也生活在陆地上。在不利的环境条件下，肺鱼进入休眠状态，这种休眠状态可以持续数月或数年，其中包括在动物身体周围形成粘液茧。我们最近发现，肺鱼茧不仅仅是一个干燥的黏液层，而是由许多活细胞组成的，包括大量的粒细胞，这些细胞是在诱导睡眠过程中，一层又一层表皮脱落时从皮肤转移过来的。茧的形成与极端的促炎状态有关，典型的促炎细胞因子和抗菌肽的高表达水平以及粒细胞从循环中大量流入。茧是如何形成的，粘膜炎症是如何引起的，目前还不清楚。这项提议的重点是在最近测序的肺鱼基因组中发现的一种新的毒素样分子。这种毒素与霍乱弧菌毒素（CtxA）相似，在系统发育上与箱水母和其他无脊椎动物毒素有关。我们假设Protop-CtxA是在进化史中通过原核生物向肺鱼的水平基因转移获得的。初步结果表明，Protop-CtxA在肺鱼发育过程中起重要作用，并在真皮干细胞中稳定表达。利用显微镜、转录组学、流式细胞术、体外抗菌和杀虫试验以及体内实验，将确定这种毒素的生物学功能。该项目由共生、感染和免疫以及促进竞争研究的既定计划（EPSCoR）共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Organismal adaptations to extreme environmental changes often involve drastic changes at mucosal barrier tissues. African lungfish can survive long drought periods via a physiological process called estivation. Estivation involves drastic remodeling of the skin and shedding of epidermal layers to form a cocoon. Previous work by our group demonstrated that the lungfish cocoon is a living tissue that contains many immune cells that trap bacteria, defending the lungfish body from pathogen invasion during a vulnerable, dormant state. Forming the cocoon, therefore involves self-inflicting mucosal inflammation but the molecular mechanisms underlaying cocoon formation are not understood. This proposal investigates a novel molecule called Protop-CtxA which is a toxin encoded in the lungfish genome. This toxin appears to have been acquired via horizontal gene transfer from bacteria or viruses. Protop-CtxA expression is highly up-regulated in the lungfish skin and cocoon upon estivation. We hypothesize that this molecule is sufficient to break down skin integrity in estivating lungfish and form in this way the cocoon. The goal of this work is to investigate which cells produce Protop-CtxA in free-swimming and estivating lungfish and to determine the inflammatory, antimicrobial and insecticidal functions of Protop-CtxA. The proposed work will advance our knowledge on extreme physiological adaptations of vertebrate animals and may result in many pharmaceutical and agricultural applications due to the bioactivity of this toxin. Broader impacts include outreach activities such as a lungfish exhibit in the Albuquerque Natural History Museum in Spanish and English and a radio podcast for the Children Hour at KUNM.The African lungfish (Protopterus sp.) lives in both water and land. Under unfavorable environmental conditions, lungfish enter estivation, a dormant state that can last for months or years and that involves the formation of a mucus cocoon that surrounds the animal body. We recently discovered that the lungfish cocoon is not just a dry mucus layer but is formed by many living cells including large numbers of granulocytes that transmigrate from the skin when layer after layer of epidermis is shed during the induction of estivation. Cocoon formation is associated with an extreme pro-inflammatory state with high expression levels of canonical pro-inflammatory cytokines and antimicrobial peptides and a large influx of granulocytes from circulation. How the cocoon is formed and mucosal inflammation instigated is not well understood. This proposal focuses on a novel toxin-like molecule discovered in the recently sequenced lungfish genome. This toxin, similar to Vibrio cholera toxin (CtxA), is phylogenetically related to box jellies and other invertebrate toxins. We hypothesize that Protop-CtxA was acquired via horizontal gene transfer from prokaryotes to lungfish during evolutionary history. Preliminary results indicate that Protop-CtxA, is very important during lungfish estivation and that dermal stem cells express it at the steady state. Using microscopy, transcriptomics, flow cytometry, in vitro antimicrobial and insecticidal assays and in vivo estivation experiments, the biological function of this toxin will be determined. This project is jointly funded by Symbiosis, Infection, and Immunity, and the Established Program to Stimulate Competitive Research (EPSCoR).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.