Molecular mechanisms underlying the emergence of polygenetic antifungal drug resistance in msh2 mismatch repair mutants of Cryptococcus

Fungal infections are common life-threatening diseases amongst immunodeficient individuals. Invasive fungal disease is commonly treated with an azole antifungal agent, resulting in selection pressure and the emergence of drug resistance. Antifungal resistance is associated with higher mortality rates and treatment failure, making the current clinical management of fungal disease very challenging. Clinical isolates from a variety of fungi have been shown to contain mutations in the MSH2 gene, encoding a component of the DNA mismatch repair pathway. Mutation of MSH2 results in an elevated mutation rate that can increase the opportunity for selectively advantageous mutations to occur, accelerating the development of antifungal resistance.To characterize the molecular mechanisms causing the microevolutionary emergence of antifungal resistance in msh2 mismatch repair mutants of Cryptococcus neoformans.The mechanisms resulting in the emergence of antifungal resistance were investigated using WGS, characterization of deletion mutants and measuring ploidy changes.The genomes of resistant strains did not possess mutations in ERG11 or other genes of the ergosterol biosynthesis pathway. Antifungal resistance was due to small contributions from mutations in many genes. MSH2 does not directly affect ploidy changes.This study provides evidence that resistance to fluconazole can evolve independently of ERG11 mutations. A common microevolutionary route to the emergence of antifungal resistance involves the accumulation of mutations that alter stress signalling, cellular efflux, membrane trafficking, epigenetic modification and aneuploidy. This complex pattern of microevolution highlights the significant challenges posed both to diagnosis and treatment of drug-resistant fungal pathogens.