Evolution of Cryptococcus neoformans Strains from Patients with HIV/AIDS

Duke University, Durham NC

Investigators

Abstract

ABSTRACT Cryptococcus neoformans is the most common and deadly fungal pathogen of the central nervous system in HIV/AIDS patients worldwide, causing hundreds of thousands of cryptococcal meningitis (CM) cases each year. With the profound loss of CD4+ immune cells and weakened blood-brain barrier, C. neoformans spreads efficiently from the lung to the brain and is difficult to treat. Clinicians lack the critical diagnostic information to identify highly pathogenic yeast isolates and successfully treat their patients because the factors that influence CM disease severity in humans are largely unknown – and no new antifungal agents have been developed in over 25 years. Preliminary data show clearly that C. neoformans isolates differ in their pathogenicity, and isolates from clinical – but not environmental – settings are associated with high CM disease severity in animal models. We will address the critical need to determine the underlying mechanisms responsible for C. neoformans pathogenicity, by defining and characterizing the evolving population of diverse isolates in both environmental and clinical settings. Our comprehensive platform of phenotyping, comparative genomics, and genome-wide association studies will enable genotype-to-phenotype analyses, focusing on virulence traits: (1) a collection/repository of 3000+ global C. neoformans isolates with sequenced genomes; (2) a high-throughput robotic system for large-scale phenotypic analyses; (3) phenotypic arraying for in vivo relevant phenotypes that can be linked to genomic data and specific clinical outcomes; (4) robust animal models (i.e. zebrafish for screening large numbers of yeast isolates; murine for brain and lung disease assessment through yeast census and immune responses; and rabbit for CNS disease validation); (5) a genetics system that facilitates multiple analyses and genetic crosses for future QTL-based studies, as well as robust genetic and molecular biology methodologies to dial down on targeted genes of interest; (6) metadata on clinical de-identified human data, as well as on isolates for yeast burden during infection and impact on mortality; and (7) defined in vivo yeast transcriptomics from isolates from human CSF to link in vitro and in vivo animal studies with human disease. Aim 1 will determine the genetic variants associated with virulence by systematic, comparative analyses of clinical and environmental isolates. This will answer the question of what turns an environmental strain that does not cause disease into a clinical strain. Aim 2 will identify and validate genetic variants associated with aggressive clinical isolates that enhance human infection. This will address the question of what causes increased aggressiveness in a clinical strain. These components will identify genes critical for C. neoformans production of aggressive disease and for in vivo survival/fitness in animal models and humans. enable us to link important disease traits with specific genomic features, in order to predict strain virulence based on genotype. Our data should lead to development of new therapeutic strategies that reduce disease severity and improve outcomes in HIV/AIDS patients. Finally, this study will create a substantial infrastructure addition to a library of well- characterized wild-type strains for use by the entire cryptococcal community for future pathobiology work.