Abstract
Cryptococcus neoformans is a human fungal pathogen that can cause fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug administered to treat cryptococcosis. When exposed to the inhibitory concentration of FLC, C. neoformans exhibits heteroresistance where a small subpopulation of cells develops into FLC-resistant colonies. FLC-resistant cells are aneuploids with regard to specific beneficial chromosomal regions. Factors underlying the potential for only certain C. neoformans cells in a genetically isogenic population to become FLC-resistant are unknown. In this study, we systematically examine the heterogeneous response of C. neoformans to FLC at a colony and individual cell level. We find that the heterogeneity in response to FLC is reflected by variable diminishment of the ergosterol at the plasma membrane. A population of C. neoformans spread on a semi-solid medium displays two types of outcomes following FLC exposure. The first outcome is colonies consisting of non-resistant cells (survivors). The size of colonies consisting of survivors ranges from a few cells to visible colonies, which reflects intrinsic phenotypic heterogeneity of the C. neoformans population. The second outcome is FLC-resistant cells forming colonies of sizes significantly larger as compared to colonies made of survivors. We propose a model that describes how a distribution of these types of cellular responses within a population changes depending on FLC concentration and factors that influence the rate of cellular growth including temperature, media type, growth phase, and the age of cells. Our findings highlight a complex nature of the response to a fungistatic drug and provide insights that may help to optimize FLC therapy.
Highlights
Single-cell clonal populations are made up of genetically homogeneous cells, but genetic identity does not necessarily translate to phenotypic similarity
An important question to consider when aiming at identifying a drug therapy that does not allow for resistance or recurrence is: Why are some genetically identical cells able to proliferate when challenged with a drug while others are inhibited? A better understanding of the intrinsic, phenotypic heterogeneity of cell populations is crucial to improve our current therapeutic approaches
Two non-exclusive possibilities may explain the heterogeneity of the response to FLC: (1) The degree of FLC-triggered depletion of ergosterol may vary from cell to cell or (2) The degree to which a cell is affected by a specific diminishment of ergosterol may vary from cell to cell
Summary
Single-cell clonal populations are made up of genetically homogeneous cells, but genetic identity does not necessarily translate to phenotypic similarity. Of particular importance is the heterogeneous response of pathogens to drug treatment, especially in the context of the development of drug resistance as it constitutes a major barrier to effective therapy. Drug therapies that aim at eliminating a certain cell type (i.e., microbial pathogens, cancer cells) often overlook the phenotypic heterogeneity that exists within the targeted cell population and its potential to promote survival during treatment. Persisters are thought to remain throughout infection; they do not make up the population of truly resistant cells, but may contribute to the formation of resistant cells through the ability to survive in the presence of the drug. An important question to consider when aiming at identifying a drug therapy that does not allow for resistance or recurrence is: Why are some genetically identical cells able to proliferate when challenged with a drug while others are inhibited? An important question to consider when aiming at identifying a drug therapy that does not allow for resistance or recurrence is: Why are some genetically identical cells able to proliferate when challenged with a drug while others are inhibited? A better understanding of the intrinsic, phenotypic heterogeneity of cell populations is crucial to improve our current therapeutic approaches
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