Candida albicans exhibits distinct cytoprotective responses to anti-fungal drugs that facilitate the evolution of drug resistance

Abstract

We developed a modified protocol for nanolitre droplet-based single cell sequencing appropriate for fungal settings, and used it to transcriptionally profiled several thousands cells from a prototrophic Candida albicans population and several drug exposed colonies (incl. fluconazole, caspofungin and nystatin). Thousands of cells from each colony were profiled both at early and late time points post-treatment in order to infer survival trajectories from initial drug tolerance to drug resistance. We find that prototrophic C. albicans populations differentially and stochastically express cytoprotective epigenetic programs. For all drugs, there is evidence that tolerant individuals partition into distinct subpopulations, each with a unique survival strategy involving different regulatory programs. These responses are weakly related to changes in morphology (shift from white to opaque forms, or shift from yeast to filamentous forms). In turn, those subpopulations that successfully reach resistance each have a distinct multivariate epigenetic response that coordinates the expression of efflux pumps, chaperones, transport mechanisms, and cell wall maintenance. Live cell fluorescent imaging was used to validate predictions of which molecular responses most often led to survival after drug exposure. Together our findings provide evidence that C. albicans has a robust toolkit of short-term epigenetic cytoprotective responses designed to "buy time" and increase the chance of acquiring long-term resistance.

Candida albicans exhibits heterogeneous and adaptive cytoprotective responses to anti-fungal compounds

“…We then constructed a nanoliter droplet-based system modified from Macosko et al using homemade components as described by Boogeshagi et al (Booeshaghi et al, 2019) to reduce the cost of both the device and each population assay. The profiles reported here combine our preliminary effort (Bettauer et al, 2020) with two additional batches of samples with complementary data and analyses to provide better power to examine the technical and biological efficacy of our system. C. albicans populations were grown in rich media alone (untreated, UT) or with an antifungal compound: fluconazole (FCZ), caspofungin (CSP), or rapamycin (RAPA) (Figure 1A -figure supplement 1A).…”

“…Only two high-throughout (10 3 to 10 4 cells) fungal transcriptome studies have been conducted in S. cerevisiae including Jackson and colleagues who profiled ~40K S. cerevisiae cells with the commercial Chromium (10X Inc.) system (Dohn et al, 2022;Jackson et al, 2020;Jariani et al, 2020). Our previous effort was the first to profile C. albicans with sc-technologies (Bettauer et al, 2020); in this study we extend and refine the data and analysis that was generated using our fungal nanoliter droplet-based assay (DROPseq), modified from the original system presented by . Our system addresses technical challenges that arise in the fungal setting and provides a flexible cost-effective solution.…”

Chromatin Structure and Drug Resistance in Candida spp.

“…One evolutionary advantage to phenotypic resistance is that, theoretically, it can ‘buy time’, which would provide the cell with an opportunity to acquire genetic lesions that confer true resistance, thus maximizing an organism’s survival. Indeed, the adaptation to anti-fungal drugs might be initially attributed to a phenotypic switch to a less drug-susceptible state, which subsequently leads to acquired resistance ( Figure 2 ) [ 85 ]. Although experimental evidence is currently lacking to support this hypothesis in Candida spp., such a combined epigenetic and genetic model of acquired drug resistance is clearly evident in tumor cells [ 86 ].…”

“…The most direct way to identify the molecular underpinnings of phenotypic resistance is through single-cell analysis, in order to characterize population heterogeneity at the level of metabolism, physiology, and transcription [ 89 ]. One such study has employed DROPseq technology [ 90 ] to monitor the single-cell transcriptome of C. albicans when exposed to anti-fungal drugs at concentrations below the MIC [ 85 ]. These authors found that the population divided into two distinct groups after 48 h, due primarily to mounting different expression profiles of stress response genes.…”

“…These data suggest that, upon anti-fungal stress, C. albicans cells explore different epigenetic landscapes, potentially to maximize survival. Furthermore, the authors found that a sub-population of cells acquired resistance after 72 h, although it was undetermined whether this was due to genetic, or possibly epigenetic, alterations [ 85 ].…”