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Ploidy change as a rapid mechanism of adaptation

Final Report Summary - RAPLODAPT (Ploidy change as a rapid mechanism of adaptation)

The study of responses to antimicrobial drugs is critical to our ability to treat the increasing burden of infectious diseases. Fungi are particularly challenging pathogens because they and their human hosts have similar (eukaryotic) biochemistry. This means that the arsenal of antifungal agents that can kill fungi while not causing problems in humans is limited. Classically drug resistance is another reason for the difficulty in treating infections. Drug resistance enables all cells in an isolate to grow at high drug concentrations, usually because of a single genetic mutation. Yet, fungal drug resistance does not explain the all of the difficulty in treating fungal infections. Our research revealed that antifungal tolerance is a missing link in explaining the failures in treating infections with Candida albicans, the most common fungal pathogen. Antifungal tolerance, the ability of some cells in the population to grow slowly in the presence of high drug concentrations, is seen to different degrees in different patient isolates and we found that patients infected with highly tolerance isolates are more likely to have prolonged infections that are not cleared by a single course of therapy with fluconazole, the most widely used antifungal drug. We found that some types of antifungal tolerance are due to changes in chromosome number (aneuploidy) within the infecting fungus, and that this aneuploidy allows cells to grow faster in an antifungal drug (and sometimes in more than one drug). We also found, when used in combination with fluconazole, inhibitors of cellular processes, including pathways that respond to nutrient status, heat stress, calcium ions and physiological signaling molecules, that are able to eliminate tolerance and to increase the ability of fluconazole to kill fungal cells, and to improve the survival of animals infected with a fungal pathogen that has high levels of tolerance. Together, these studies suggest that monitoring antifungal tolerance of infecting isolates could improve clinical decisions for the treatment of fungal infections. Furthermore, it could guide clinicians in the use of combinations of antifungals with inhibitors that eliminate tolerance.
To perform these experiments, we developed several novel assays of fungal growth and drug responses, several of them based on time-lapse technologies, which provide a dynamic report of how different cells respond to drug over extended time periods. We also utilized novel fluorescently labeled azole antifungal drugs, produced by Prof. M. Fridman for this project, to determine the intracellular localization of the drugs. Directing the drug to localize to the same organelle as the enzyme that it inhibits increased drug efficacy by two orders of magnitude. We also exploited our discovery of C. albicans haploid state to produce a collection of over half a million isolates, each carrying a different mutation in the genome, using a modified maize transposon. This collection allowed us, with the help of machine learning algorithms, to predict the degree to which each gene in the C. albicans genome is essential for growth under lab conditions. We are currently using this collection to study the full complement of genes necessary for C. albicans resistance and/or tolerance to commonly used antifungal drugs and drug combinations.