Deciphering the significance of pathogen genetic variation and evolution

PROJECT SUMMARY Using a combination of genetics and experimental evolution, our team seeks to understand complex epistasis with respect to genetic background in the human pathogen Cryptococcus neoformans. This yeast is an ideal platform for such investigations due to 1) its facile genetics, 2) the availability of hundreds of phylogenetically well-separated genomes, and 3) mouse infection models that recapitulate key aspects of human disease. Most studies have been performed with a reference strain, the clinical isolate H99, or in congenic derivatives (KN99a and KN99). In unpublished work, our lab has generated a gene deletion strain collection in the KN99 background and profiled it in mice and in over 100 diverse in vitro conditions. A reference strain is important for the field to be able to compare findings, but it has a major weakness: across genetic backgrounds, it is unknown whether the same genes are 1) essential for viability, 2) mediate the responses to small molecule challenges, and 3) are required for fitness in the mammalian host. In addition to being of fundamental scientific importance, answering these questions, which all relate to the larger question of complex epistasis, is important for developing effective therapies across genetic backgrounds and understanding drug resistance. Population genomic analyses have revealed that C. neoformans exists in roughly four major clades that diverged ~5 million years ago and show evidence of speciation. Over a million SNPs have been identified in a set of nearly 400 strains. Members of each clade have been identified in human infections, and there is evidence for clade- specific traits. Anecdotal studies have provided examples of strain-specific phenotypes, raising the question of what genotype-phenotype relationships are general vs. dependent on genetic background and the underlying mechanisms. Powerful new CRISPR/Cas9-based tools that we have recently developed and other technological advances now make it feasible to address this fundamental question. In this work, we will determine how Cryptococcus neoformans genetic background impacts the fundamental traits of gene essentiality (Aim 1), the role of genes in fitness in the mammalian host (Aim 2), and the fitness roles of genes under diverse environments (Aim 3). We will pursue mechanisms of the latter through experimental evolution (Aim 3).