Presentation description

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lethal meningoencephalitis in immunocompromised populations. There are an estimated 215,000 cryptococcal infections per year, with a mortality rate of 30-70% and a high prevalence in areas of limited medical infrastructure, such as Africa. Dissemination and organ seeding of C. neoformans are poorly understood. Since disseminated brain infection is the primary cause of mortality, studying these aspects of pathogenesis is crucial to developing new therapeutics. |The goal of this research is to use DNA barcoding as a means to understand the kinetics of dissemination in cryptococcosis. Using CRISPR-Cas9 technology, I inserted unique 12-nucleotide segments of DNA into the genomes of wild type C. neoformans. The barcodes are bound to an anti-fungal resistance cassette that allows for selection of fungi that successfully integrated the DNA. The barcode and resistance cassette are inserted into a genomic safe haven, ensuring no changes to fungal virulence. Through serial plating on anti-fungal media, we selected for barcode-containing fungi and confirmed insertion with Sanger sequencing. These barcoded strains can be tracked throughout infection using amplicon sequencing. We hypothesize that seeding events are rare early in infection and increase in frequency as the disease progresses. In summary, we used CRISPR-Cas9 technology to generate a pool of DNA-barcoded strains of the fungal pathogen, C. neoformans. We will use these barcoded strains in subsequent studies to define the kinetics of dissemination in cryptococcosis.