Highly Recombinant VGII Cryptococcus gattii Population Develops Clonal Outbreak Clusters through both Sexual Macroevolution and Asexual Microevolution

Abstract

An outbreak of the fungal pathogen Cryptococcus gattii began in the Pacific Northwest (PNW) in the late 1990s. This outbreak consists of three clonal subpopulations: VGIIa/major, VGIIb/minor, and VGIIc/novel. Both VGIIa and VGIIc are unique to the PNW and exhibit increased virulence. In this study, we sequenced the genomes of isolates from these three groups, as well as global isolates, and analyzed a total of 53 isolates. We found that VGIIa/b/c populations show evidence of clonal expansion in the PNW. Whole-genome sequencing provided evidence that VGIIb originated in Australia, while VGIIa may have originated in South America, and these were likely independently introduced. Additionally, the VGIIa outbreak lineage may have arisen from a less virulent clade that contained a mutation in the MSH2 ortholog, but this appears to have reverted in the VGIIa outbreak strains, suggesting that a transient mutator phenotype may have contributed to adaptation and evolution of virulence in the PNW outbreak. PNW outbreak isolates share genomic islands, both between the clonal lineages and with global isolates, indicative of sexual recombination. This suggests that VGII C. gattii has undergone sexual reproduction, either bisexual or unisexual, in multiple locales contributing to the production of novel, virulent subtypes. We also found that the genomes of two basal VGII isolates from HIV+ patients contain an introgression tract spanning three genes. Introgression substantially contributed to intra-VGII polymorphism and likely occurred through sexual reproduction with VGI. More broadly, these findings illustrate how both microevolution and sexual reproduction play central roles in the development of infectious outbreaks from avirulent or less virulent progenitors.

Cryptococcus gattii Infections

“…The WGST data showed clearly that the VGIIc genotype responsible for the most recent infections in the Pacific Northwest is genetically distinct from both VGIIa and VGIIb Vancouver Island outbreak subtypes and suggest that all three subtypes are equally distinct from each other (52). Similar findings based on multilocus sequence typing (MLST) and MLMT analyses, which have been confirmed based on WGST, have been reported recently (53)(54)(55). All studies suggest that the three subtypes have arisen as a result of recombination events that took place prior to their introduction into the Pacific Northwest.…”

Unisexual Reproduction Reverses Muller’s Ratchet

“…This laboratory validation supports the growing observational evidence that unisexual reproduction plays a role in producing virulent strains from avirulent or less virulent parents in nature (Fraser et al 2005;Byrnes et al 2010Byrnes et al , 2011. In outbreaks in the Pacific Northwest of the United States and Canada, population genomic data suggest that sexual reproduction occurred prior to clonal expansion of hypervirulent strains (Fraser et al 2005;Billmyre et al 2014). Together, this suggests that sexual reproduction may play an important role in producing and maintaining virulent pathogens.…”

“…Recent evidence has suggested that unisexual reproduction and recombination play an important role in generating virulent strains of Cryptococcus and other eukaryotic pathogens, including both fungi and parasites (Jenni et al 1986;Gaunt et al 2003;Fraser et al 2005;Akopyants et al 2009;Byrnes et al 2010Byrnes et al , 2011Wendte et al 2010;Minot et al 2012;Inbar et al 2013;Voelz et al 2013;Billmyre et al 2014). We tested whether parental strains with mutations that compromise virulence are able to undergo unisexual reproduction to produce virulent progeny.…”

Emerging issues, challenges, and changing epidemiology of fungal disease outbreaks

“…Phylogenetic analyses indicate that the VGII type likely originated in South America and underwent multiple dispersal events, but exactly how and why C gattii arrived in the PNW remains unknown. 23–25 As a result of this outbreak, Oregon and Washington began requiring physicians and laboratories to report C gattii cases to public health authorities, and laboratories began to differentiate C gattii from Cryptococcus neoformans .…”