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

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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.