The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in healthcare settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five Food and Drug Administration (FDA)-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris . To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only twofold to fivefold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1 ) and in the drug transporter CDR1 . These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more susceptible to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. IMPORTANCE: The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated 8-hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris . Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest twofold to fivefold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500-fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated 8-hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris . Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.

Biofilms formed by the human fungal pathogen Candida albicans are naturally resistant to many of the antifungal agents commonly used in the clinic. We screened a library containing 1600 clinically tested drug compounds to identify compounds that inhibit C. albicans biofilm formation. The compounds that emerged from the initial screen were validated in a secondary screen and then tested for (1) their abilities to disrupt mature biofilms and (2) for synergistic interactions with representatives of the three antifungal agents most commonly prescribed to treat Candida infections, fluconazole, amphotericin B, and caspofungin. Twenty compounds had antibiofilm activity in at least one of the secondary assays and several affected biofilms but, at the same concentration, had little or no effect on planktonic (suspension) growth of C. albicans. Two calcium channel blockers, a selective serotonin reuptake inhibitor, and an azole-based proton pump inhibitor were among the hits, suggesting that members of these three classes of drugs or their derivatives may be useful for treating C. albicans biofilm infections.

White and opaque cells of Candida albicans have the same genome but differ in gene expression patterns, metabolic profiles, and host niche preferences. We tested whether these differences (which include the differential expression of drug transporters) resulted in different sensitivities to 27 antifungal agents. The analysis was performed in two different strain backgrounds; although there was strain-to-strain variation, only terbinafine hydrochloride and caspofungin showed consistent, two-fold differences between white and opaque cells across both strains.

The human fungal pathogen Candida albicans can form biofilms on biotic and abiotic surfaces, which are inherently resistant to antifungal drugs. We screened the Chembridge Small Molecule Diversity library containing 30,000 “drug-like” small molecules and identified 45 compounds that inhibited biofilm formation. These 45 compounds were then tested for their abilities to disrupt mature biofilms and for combinatorial interactions with fluconazole, amphotericin B, and caspofungin, the three antifungal drugs most commonly prescribed to treat Candida infections. In the end, we identified one compound that moderately disrupted biofilm formation on its own and four compounds that moderately inhibited biofilm formation and/or moderately disrupted mature biofilms only in combination with either caspofungin or fluconazole. No combinatorial interactions were observed between the compounds and amphotericin B. As members of a diversity library, the identified compounds contain “drug-like” chemical backbones, thus even seemingly “weak hits” could represent promising chemical starting points for the development and the optimization of new classes of therapeutics designed to target Candida biofilms.

Candida albicans is a normal member of the human microbiome and an opportunistic fungal pathogen. This species undergoes several morphological transitions, and here we consider white-opaque switching. In this switching program, C. albicans reversibly alternates between two cell types, named “white” and “opaque,” each of which is normally stable across thousands of cell divisions. Although switching under most conditions is stochastic and rare, certain environmental signals or genetic manipulations can dramatically increase the rate of switching. Here, we report the identification of two new inputs which affect white-to-opaque switching rates. The first, exposure to sub-micromolar concentrations of (E,E)-farnesol, reduces white-to-opaque switching by ten-fold or more. The second input, an inferred PKA phosphorylation of residue T208 on the transcriptional regulator Efg1, increases white-to-opaque switching ten-fold. Combining these and other environmental inputs results in a variety of different switching rates, indicating that a given rate represents the integration of multiple inputs.

An unusual feature of the opportunistic pathogen C. albicans is its ability to stochastically switch between two distinct, heritable cell types called white and opaque. Here, we show that only opaque cells, in response to environmental signals, massively up-regulate a specific group of secreted proteases and peptide transporters, allowing exceptionally efficient use of proteins as sources of nitrogen. We identify the specific proteases (members of the secreted aspartyl protease ( SAP ) family) needed for opaque cells to proliferate under these conditions, and we identify four transcriptional regulators of this specialized proteolysis and uptake program. We also show that, in mixed cultures, opaque cells enable white cells to also proliferate efficiently when proteins are the sole nitrogen source. Based on these observations, we suggest that one role of white-opaque switching is to create mixed populations where the different phenotypes derived from a single genome are shared between two distinct cell types. Summary: The opportunistic human fungal pathogen Candida albicans switches between two distinct, heritable cell types, named “white” and “opaque.” We show that opaque cells, in response to proteins as the sole nitrogen source, up-regulate a specialized program, including specific secreted aspartyl proteases and peptide transporters. We demonstrate that, in mixed cultures, opaque cells enable white cells to respond and proliferate more efficiently under these conditions. These observations suggest that white-opaque switching creates mixtures of cells where the population characteristics - which derive from a single genome - reflect the contributions of two distinct cell types. Dataset Reference Numbers: The .RAW files for both sets of Mass Spectrometry experiments have been deposited at the ProteoSAFe resource ( https://proteomics.ucsd.edu/ProteoSAFe/ ). MSP-MS experiment reference number: MSV000085279. For reviewer access use login “MSV000085279_reviewer” and password “candidamspms”. Proteomics experiment reference number: MSV000085283. For reviewer access use login “MSV000085283_reviewer” and password “candidaprot”.