Genomic Analysis of the Population Structure and Virulence of the Human Pathogen, Cryptococcus Neoformans Var. Grubii

Abstract

This study examined a large cohort of clinical and environmental strains of Cryptococcus neoformans var. grubii to better understand its population structure and identify potential markers significantly associated with clinical isolates or increased virulence capacity. C. neoformans var. grubii is an opportunistic fungal pathogen infecting nearly 1 million patients a year, mostly immunocompromised individuals. Previous work using multi-locus sequence typing analysis identified three sublineages: VNI, VNII, and VNB. Unlike the other two sublineages, the VNB sublineage is constrained to sub-Saharan Africa, exhibits high genetic variability, and encompasses a mix of environmental and clinical isolates. Nearly 400 diverse isolates were selected for sequencing and SNP genotype data were generated and provided as input to the STRUCTURE and smartPCA software to further clarify the population structure. This population analysis revealed a split in the VNB sublineage along with the presence of hybrid and introgressed strains. These genotype data and population information were used with the GEMMA software to identify potential markers significantly associated with clinical strains. GWAS identified twelve markers significantly associated with clinical strains, several within known virulence genes. Additional GWAS analysis utilized phenotypic characterization data for oxidative stress, fluconazole drug resistance, and melanization along with the genotype data. This revealed that three clinical strains harbored significantly associated mutations in BZP4 and exhibited decreased melanin production similar to the melanin-deficient Lac1 deletion strain. These data highlight the utility of GWAS in the analysis of complex traits of pathogenic fungi and provide new markers for further virulence studies.