Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis.

BACKGROUND Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) - a resistant subpopulation within a susceptible strain - is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. METHODS A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. RESULTS Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. CONCLUSION Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. FUNDING This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship

A Novel Assay Reveals a Maturation Process during Ascospore Wall Formation

The ascospore wall of the budding yeast Saccharomyces cerevisiae consists of inner layers of similar composition to the vegetative cell wall and outer layers made of spore-specific components that confer increased stress resistance on the spore. The primary constituents of the outer spore wall are chitosan, dityrosine, and a third component termed Chi that has been identified by spectrometry but whose chemical structure is not known. The lipophilic dye monodansylpentane readily stains lipid droplets inside of newly formed ascospores but, over the course of several days, the spores become impermeable to the dye. The generation of this permeability barrier requires the chitosan layer, but not dityrosine layer, of the spore wall. Screening of a set of mutants with different outer spore wall defects reveals that impermeability to the dye requires not just the presence of chitosan, but another factor as well, possibly Chi, and suggests that the OSW2 gene product is required for synthesis of this factor. Testing of mutants that block synthesis of specific aromatic amino acids indicates that de novo synthesis of tyrosine contributes not only to formation of the dityrosine layer but to impermeability of the wall as well, suggesting a second role for aromatic amino acids in spore wall synthesis