While mechanisms of resistance to major antifungal agents have been characterized in Candida albicans, little is known about the evolutionary trajectories during the emergence of drug resistance. Here, we examined the evolutionary dynamics of C. albicans that evolved in vitro in the presence or absence of fluconazole using the visualizing evolution in real-time (VERT) method, a novel experimental approach that facilitates the systematic isolation of adaptive mutants that arise in the population. We found an increase in the frequency of adaptive events in the presence of fluconazole compared to the no-drug controls. Analysis of the evolutionary dynamics revealed that mutations that led to increased drug resistance appeared frequently and that mutants with increased levels of resistance arose in independent lineages. Interestingly, most adaptive mutants with increased fitness in the presence of the drug did not exhibit a significant fitness decrease in the absence of the drug, supporting the idea that rapid resistance can arise from mutations in strains maintained in the population prior to exposure to the drug.The emergence of antimicrobial drug resistance in pathogens is a process of adaptive evolution, generally as a result of genetic mutations. Depending on the size of the population, the rate of mutation, and the relative fitness coefficients, the population may be heterogeneous, consisting of multiple resistant genotypes competing for expansion in a process called "clonal interference" (10, 12). Determining the evolutionary dynamics during adaptation is important for understanding the fundamental principles underlying how eukaryotic microbes evolve resistance to antimicrobial agents, a process that differs from that in bacteria because it is unlikely to involve horizontal gene transfer (1, 6).It is important to understand the frequencies with which adaptive mutants arise and expand, the evolutionary trajectories (the order of occurrence of adaptive mutations), and the potential convergence or divergence in the adaptive mechanisms between parallel populations in order to better appreciate how drug resistance can emerge in pathogens growing within the host. For example, knowledge of the frequency and order in which drug-resistant mutants arise in the population and whether the early-arising resistance mechanisms play a role in the level of drug resistance ultimately reached in the population can be used to predict the likely trajectory of a clinical infection and to develop appropriate therapeutic strategies.Candida albicans is the fourth most common cause of nosocomial infections, resulting in rates of mortality in U.S. hospitals that approach 50% (23,24,44). Emergence of resistance to almost all major antifungal agents used in treating C. albicans infections has been reported (15,22,29). Among the existing classes of antifungal drugs, azoles are the most commonly used, due to their low toxicity and their oral availability. The fact that they are fungistatic rather than fungicidal provides a clear opportunity...

Candida albicans is an important human fungal pathogen. Resistance to all major antifungal agents has been observed in clinical isolates of Candida spp. and is a major clinical challenge. The rise and expansion of drug-resistant mutants during exposure to antifungal agents occurs through a process of adaptive evolution, with potentially complex population dynamics. Understanding the population dynamics during the emergence of drug resistance is important for determining the fundamental principles of how fungal pathogens evolve for resistance. While few detailed reports that focus on the population dynamics of C. albicans currently exist, several important features on the population structure and adaptive landscape can be elucidated from existing evolutionary studies in in vivo and in vitro systems.

Candida glabrata (C glabrata) is an important yeast of industrial and medical significance. Resistance to oxidative stress is an important trait affecting its robustness as a production host or virulence as a pathogenic agent, but current understanding of resistance mechanisms is still limited in this fungus. In this study, we rapidly evolved C glabrata population to adapt to oxidative challenge (from 80mM to 350mM of H O ) through short-term adaptive laboratory evolution. Adaptive mutants were isolated from evolved populations and subjected to phenotypic and omics analyses to identify potential mechanisms of tolerance to H O . Phenotypic characterizations revealed faster detoxification of H O and ability to initiate growth at a higher concentration of the oxidant in the isolated adaptive mutants compared with the wild type. Genome resequencing and genome-wide transcriptome analysis revealed multiple genetic determinants (eg, CAGL0E01243g, CAGL0F06831g, and CAGL0C00385g) that potentially contribute to enhanced H O resistance. Subsequent experimental verification confirmed that CgCth2 (CAGL0E01243g) and CgMga2 (CAGL0F06831g) are important in C glabrata tolerance to oxidative stress. Transcriptome profiling of adaptive mutants and bioinformatic analysis suggest that NADPH regeneration, modulation of membrane composition, cell wall remodeling, and/or global regulatory changes are involved in C glabrata tolerance to H O .

Candida glabrata is a human commensal and an opportunistic human fungal pathogen. It is more closely related to the model yeast Saccharomyces cerevisiae than other Candida spp. Compared with S. cerevisiae, C. glabrata exhibits higher innate tolerance to various environmental stressors, including hyperthermal stress. Here we investigate the molecular mechanisms of C. glabrata adaptation to heat stress via adaptive laboratory evolution. We show that all parallel evolved populations readily adapt to hyperthermal challenge (from 47 °C to 50 °C) and exhibit convergence in evolved phenotypes with extensive cross-tolerance to various other environmental stressors such as oxidants, acids, and alcohols. Genome resequencing identified fixation of mutations in CgSTE11 in all parallel evolved populations. The CgSTE11 homolog in S. cerevisiae plays crucial roles in various mitogen-activated protein kinase (MAPK) signaling pathways, but its role is less understood in C. glabrata. Subsequent verification confirmed that CgSTE11 is important in hyperthermal tolerance and the observed extensive cross-tolerance to other environmental stressors. These results support the hypothesis that CgSTE11 mediates cross-talks between MAPK signaling pathways in C. glabrata in response to environmental challenges.

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