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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.

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.

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.

An unusual feature of the opportunistic pathogen Candida albicans is its ability to switch stochastically between two distinct, heritable cell types called white and opaque. Here, we show that only opaque cells, in response to environmental signals, massively upregulate 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.

The fungal species Candida albicans is both a member of the human microbiome and a fungal pathogen. C. albicans undergoes several different morphological transitions, including one called white-opaque switching. Here, cells reversibly switch between two states, "white" and "opaque," and each state is heritable through many cell generations. Each cell type has a distinct cellular and colony morphology and they differ in many other properties including mating, nutritional specialization, and interactions with the innate immune system. Previous genetic screens to gain insight into white-opaque switching have focused on certain classes of genes (for example transcriptional regulators or chromatin modifying enzymes). In this paper, we examined 172 deletion mutants covering a broad range of cell functions. We identified 28 deletion mutants with at least a fivefold effect on switching frequencies; these cover a wide variety of functions ranging from membrane sensors to kinases to proteins of unknown function. In agreement with previous reports, we found that components of the pheromone signaling cascade affect white-to-opaque switching; however, our results suggest that the major effect of Cek1 on white-opaque switching occurs through the cell wall damage response pathway. Most of the genes we identified have not been previously implicated in white-opaque switching and serve as entry points to understand new aspects of this morphological transition.

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.

Biofilms formed by the fungal pathogen Candida albicans are resistant to many of the antifungal agents commonly used in the clinic. Previous reports suggest that protease inhibitors, specifically inhibitors of aspartyl proteases, could be effective antibiofilm agents. We screened three protease inhibitor libraries, containing a total of 80 compounds for the abilities to prevent C. albicans biofilm formation and to disrupt mature biofilms. The compounds were screened individually and in the presence of subinhibitory concentrations of the most commonly prescribed antifungal agents for Candida infections: fluconazole, amphotericin B, or caspofungin. Although few of the compounds affected biofilms on their own, seven aspartyl protease inhibitors inhibited biofilm formation when combined with amphotericin B or caspofungin. Furthermore, nine aspartyl protease inhibitors disrupted mature biofilms when combined with caspofungin. These results suggest that the combination of standard antifungal agents together with specific protease inhibitors may be useful in the prevention and treatment of C. albicans biofilm infections.

Differentiated cell types often retain their characteristics through many rounds of cell division. A simple example is found in Candida albicans, a member of the human microbiota and also the most prevalent fungal pathogen of humans; here, two distinct cell types (white and opaque) exist, and each one retains its specialized properties across many cell divisions. Switching between the two cell types is rare in standard laboratory medium (2% glucose) but can be increased by signals in the environment, for example, certain sugars. When these signals are removed, switching ceases and cells remain in their present state, which is faithfully passed on through many generations of daughter cells. Here, using an automated flow cytometry assay to monitor white-opaque switching over 96 different sugar concentrations, we observed a wide range of opaque-to-white switching that varied continuously across different sugar compositions of the medium. By also measuring white cell proliferation rates under each condition, we found that both opaque-to-white switching and selective white cell proliferation are required for entire populations to shift from opaque to white. Moreover, the switching frequency correlates with the preference of the resulting cell type for the growth medium; that is, the switching is adjusted to increase in environments that favor white cell proliferation. The widely adjustable, all-or-none nature of the switch, combined with the long-term heritability of each state, is distinct from conventional forms of gene regulation, and we propose that it represents a strategy used by C. albicans to efficiently colonize different niches of its human host.

Candida albicans is a normal member of the human microbiota that asymptomatically colonizes healthy individuals, however it is also an opportunistic pathogen that can cause severe infections, especially in immunocompromised individuals. The medical impact of C. albicans depends, in part, on its ability to form biofilms, communities of adhered cells encased in an extracellular matrix. Biofilms can form on both biotic and abiotic surfaces, such as tissues and implanted medical devices. Once formed, biofilms are highly resistant to antifungal agents and the host immune system, and can act as a protected reservoir to seed disseminated infections. Here, we present several in vitro biofilm protocols, including protocols that are optimized for high-throughput screening of mutant libraries and antifungal compounds. We also present protocols to examine specific stages of biofilm development and protocols to evaluate interspecies biofilms that C. albicans forms with interacting microbial partners. © 2018 by John Wiley & Sons, Inc.

Candida albicans is among the most prevalent fungal species of the human microbiota and asymptomatically colonizes healthy individuals. However, it is also an opportunistic pathogen that can cause severe, and often fatal, bloodstream infections. The medical impact of C. albicans typically depends on its ability to form biofilms, which are closely packed communities of cells that attach to surfaces, such as tissues and implanted medical devices. In this Review, we provide an overview of the processes involved in the formation of C. albicans biofilms and discuss the core transcriptional network that regulates biofilm development. We also consider some of the advantages that biofilms provide to C. albicans in comparison with planktonic growth and explore polymicrobial biofilms that are formed by C. albicans and certain bacterial species.

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, 2-fold differences between white and opaque cells across both strains.

Candida albicans biofilms have a significant medical impact due to their rapid growth on implanted medical devices, their resistance to antifungal drugs, and their ability to seed disseminated infections. Biofilm assays performed in vitro allow for rapid, high-throughput screening of gene deletion libraries or antifungal compounds and typically serve as precursors to in vivo studies. Here, we compile and discuss the protocols for several recently published C. albicansin vitro biofilm assays. We also describe improved versions of these protocols as well as novel in vitro assays. Finally, we consider some of the advantages and disadvantages of these different types of assays.

UnlabelledCandida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation.ImportanceBiofilm formation by the opportunistic fungal pathogen C. albicans is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by C. albicans biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during C. albicans biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from C. albicans and also from additional pathogenic Candida species. Furthermore, SAP5 and SAP6 deletions confirm that both proteases are required for proper biofilm development in vitro and in vivo We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of Candida biofilm-associated infections.

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology.

UnlabelledThe human commensal and opportunistic pathogen Candida albicans can switch between two distinct, heritable cell types, named "white" and "opaque," which differ in morphology, mating abilities, and metabolic preferences and in their interactions with the host immune system. Previous studies revealed a highly interconnected group of transcriptional regulators that control switching between the two cell types. Here, we identify Ssn6, the C. albicans functional homolog of the Saccharomyces cerevisiae transcriptional corepressor Cyc8, as a new regulator of white-opaque switching. In A: or α mating type strains, deletion of SSN6 results in mass switching from the white to the opaque cell type. Transcriptional profiling of ssn6 deletion mutant strains reveals that Ssn6 represses part of the opaque cell transcriptional program in white cells and the majority of the white cell transcriptional program in opaque cells. Genome-wide chromatin immunoprecipitation experiments demonstrate that Ssn6 is tightly integrated into the opaque cell regulatory circuit and that the positions to which it is bound across the genome strongly overlap those bound by Wor1 and Wor2, previously identified regulators of white-opaque switching. This work reveals the next layer in the white-opaque transcriptional circuitry by integrating a transcriptional regulator that does not bind DNA directly but instead associates with specific combinations of DNA-bound transcriptional regulators.ImportanceThe most common fungal pathogen of humans, C. albicans, undergoes several distinct morphological transitions during interactions with its host. One such transition, between cell types named "white" and "opaque," is regulated in an epigenetic manner, in the sense that changes in gene expression are heritably maintained without any modification of the primary genomic DNA sequence. Prior studies revealed a highly interconnected network of sequence-specific DNA-binding proteins that control this switch. We report the identification of Ssn6, which defines an additional layer of transcriptional regulation that is critical for this heritable switch. Ssn6 is necessary to maintain the white cell type and to properly express the opaque cell transcriptional program. Ssn6 does not bind DNA directly but rather associates with specific combinations of DNA-bound transcriptional regulators to control the switch. This work is significant because it reveals a new level of regulation of an important epigenetic switch in the predominant fungal pathogen of humans.

Chromatin immunoprecipitation experiments are critical to investigating the interactions between DNA and a wide range of nuclear proteins within a cell or biological sample. In this chapter we outline an optimized protocol for genome-wide chromatin immunoprecipitation that has been used successfully for several distinct morphological forms of numerous yeast species, and include an optimized method for amplification of chromatin immunoprecipitated DNA samples and hybridization to a high-density oligonucleotide tiling microarray. We also provide detailed suggestions on how to analyze the complex data obtained from these experiments.

WOPR-domain proteins are found throughout the fungal kingdom where they function as master regulators of cell morphology and pathogenesis. Genetic and biochemical experiments previously demonstrated that these proteins bind to specific DNA sequences and thereby regulate transcription. However, their primary sequence showed no relationship to any known DNA-binding domain, and the basis for their ability to recognize DNA sequences remained unknown. Here, we describe the 2.6-Å crystal structure of a WOPR domain in complex with its preferred DNA sequence. The structure reveals that two highly conserved regions, separated by an unconserved linker, form an interdigitated β-sheet that is tilted into the major groove of DNA. Although the main interaction surface is in the major groove, the highest-affinity interactions occur in the minor groove, primarily through a deeply penetrating arginine residue. The structure reveals a new, unanticipated mechanism by which proteins can recognize specific sequences of DNA.

The duplication of transcription regulators can elicit major regulatory network rearrangements over evolutionary timescales. However, few examples of duplications resulting in gene network expansions are understood in molecular detail. Here we show that four Candida albicans transcription regulators that arose by successive duplications have differentiated from one another by acquiring different intrinsic DNA-binding specificities, different preferences for half-site spacing, and different associations with cofactors. The combination of these three mechanisms resulted in each of the four regulators controlling a distinct set of target genes, which likely contributed to the adaption of this fungus to its human host. Our results illustrate how successive duplications and diversification of an ancestral transcription regulator can underlie major changes in an organism's regulatory circuitry.

The human fungal pathogen Candida albicans can switch between two phenotypic cell types, termed 'white' and 'opaque'. Both cell types are heritable for many generations, and the switch between the two types occurs epigenetically, that is, without a change in the primary DNA sequence of the genome. Previous work identified six key transcriptional regulators important for white-opaque switching: Wor1, Wor2, Wor3, Czf1, Efg1, and Ahr1. In this work, we describe the structure of the transcriptional network that specifies the white and opaque cell types and governs the ability to switch between them. In particular, we use a combination of genome-wide chromatin immunoprecipitation, gene expression profiling, and microfluidics-based DNA binding experiments to determine the direct and indirect regulatory interactions that form the switch network. The six regulators are arranged together in a complex, interlocking network with many seemingly redundant and overlapping connections. We propose that the structure (or topology) of this network is responsible for the epigenetic maintenance of the white and opaque states, the switching between them, and the specialized properties of each state.

The human fungal pathogen Candida albicans resides asymptomatically in the gut of most healthy people but causes serious invasive diseases in immunocompromised patients. Many C. albicans strains have the ability to stochastically switch between distinct white and opaque cell types, but it is not known with certainty what role this switching plays in the physiology of the organism. Here, we report a previously undescribed difference between white and opaque cells, namely their interaction with host phagocytic cells. We show that both Drosophila hemocyte-derived S2 cells and mouse macrophage-derived RAW264.7 cells preferentially phagocytose white cells over opaque cells. This difference is seen both in the overall percentage of cultured cells that phagocytose white versus opaque C. albicans and in the average number of C. albicans taken up by each phagocytic cell. We conclude that susceptibility to phagocytosis by cells of the innate immune system is an important distinction between white and opaque C. albicans, and propose that one role of switching from the prevalent white form into the rarer opaque form may be to allow C. albicans to escape phagocytosis.