MCA: Immune gene manipulation to understand antifungal immunity in frogs

The University Of Central Florida Board Of Trustees, Orlando FL

Investigators

Abstract

The rise of infectious diseases in humans and animals highlights the need to better understand how organisms use their immune systems to fight off pathogens. Frogs are declining worldwide because of the infectious fungal disease chytridiomycosis, and defining how their immune systems function is critical for developing mitigation strategies to prevent ongoing extinctions. This project provides protected research time and training for a mid-career investigator to use genetic engineering on laboratory frogs to alter their immune system genes. The project will identify how manipulating the genome of a model amphibian alters its ability to cope with infection by the highly destructive fungal pathogen Batrachochytrium dendrobatidis (Bd). By changing the genes from susceptible to resistant forms, then measuring whether they improve the survival of genetically engineered frogs, this project serves as a test of whether genome manipulation can be an effective tool in the fight against the ongoing global loss of biodiversity. This project will develop new tools for using gene editing to modify immune genes across different frog species, providing an important resource for amphibian researchers and conservation biologists. The project will also offer training to diverse early career scientists through support of undergraduate and graduate research, and will address negative perceptions of gene editing by engaging undergraduate students and the general public in research-related activities. A core dogma of immunology is that major histocompatibility complex (MHC) presentation of pathogens to T cell receptors is required to initiate adaptive immune responses. MHC genetic variation is associated with Bd resistance across a range of amphibians, but evidence to date is correlative. The central hypothesis of this proposal is that expression of specific MHC genetic variants determines fungal disease outcomes in amphibians. The well-characterized, Bd-susceptible model frog Xenopus tropicalis will be used as proof of principle for assessing the benefits and costs of genetically modifying MHC genes. This proposal will provide protected time for the PI to develop new skills in genome manipulation and immunological techniques in collaboration with Xenopus immunologists via three specific aims: (1) test the isolated effect of altering MHC sequences from susceptible to resistant forms using CRISPR/Cas9 genome editing; (2) test the effect of altering MHC expression on Bd resistance using promoter-based transgene expression of candidate MHC resistance alleles; and (3) measure the fitness consequences of MHC genetic modification via experimental fungal exposures testing Bd susceptibility and immune function, and captive breeding to quantify reproductive fitness in genetically altered animals. Together, these three aims will determine whether transplanting elements of the immune system between species elucidates mechanisms of resistance and whether this technique could generate exogenous pathogen resistance. 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.