Distinct pathways of adaptive evolution inCryptococcus neoformansreveal a point mutation in adenylate cyclase with drastic tradeoffs for pathogenicity

Abstract

Pathogenic fungi populate a wide range of environments and infect a diversity of host species. Despite this substantial biological flexibility, the impact of interactions between fungi and their hosts on the evolution of pathogenicity remains unclear. We studied how repeated interactions between the fungus Cryptococcus neoformans and relevant environmental and mammalian host cells, amoeba and mouse macrophages, shape the evolution of this model fungal pathogen. First, using a collection of clinical and environmental isolates of C. neoformans, we characterized a range of survival phenotypes for these strains when exposed to host cells of different species. We then performed serial passages of an environmentally isolated C. neoformans strain through either amoeba or macrophages for ~75 generations to observe how these interactions select for improved replication within hosts. In an adapted population, we identified a single point mutation in the adenylate cyclase gene, CAC1, that swept to fixation and confers a strong competitive advantage for growth inside of macrophages. Strikingly, this growth advantage in macrophages is inversely correlated with disease severity during mouse infections, suggesting that adaptations to specific host niches can markedly reduce the pathogenicity of these fungi. These results raise intriguing questions about the influence of cAMP signaling on pathogenicity and highlight the role of seemingly small adaptive changes in promoting fundamental shifts in the intracellular behavior and virulence of these important human pathogens.

Distinct routes to thermotolerance in the fungal pathogenCryptococcus neoformans

“…A chromosome scale assembly of the parental Ftc239-1 strain was drafted largely as previously published 34 . Briefly, fast5 files from Nanopore sequencing were re-basecalled using guppy-gpu (v6.4.2) and the resulting fastq files were assembled using Canu (v 2.1.1) with an estimated genome size of 20 megabases and default settings 38 .…”

“…Colonies were genotyped by colony PCR following previously published protocols 34 . Genomic sequences were amplified from gDNA extracted from candidate colonies and integration of the drug cassette at the SH2 locus was confirmed by Sanger sequencing.…”

“…PCRs were also performed to ensure that no strains had integrated the gRNA or CAS9 constructs. All primer sequences for generating CRISPR reagents and genotyping mutants have been previously published 34 .…”