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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 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.

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.

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.

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.

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.

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.

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.

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.

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.

The human fungal pathogen Candida albicans undergoes a switch between two distinct cell types, referred to as white and opaque. These cell types differ in cell and colony morphologies, ability to mate, metabolic preferences, preferred niches in the host, and interactions with the host innate immune system. Both cell types are stable through many generations; switching between them is rare, stochastic, and occurs without any known changes in the primary sequence of the genome; thus the switch is epigenetic. When I started my work, the circuit regulating this switch had just been identified and recently published results suggested that the innate immune system recognized the two cell types differently. My work determined that both the Drosophila hemocyte-derived S2 cell line and the mouse macrophage-derived RAW264.7 cell lines preferentially phagocytose white cells over opaque cells. This difference is observed for both the percentage of S2 or RAW cells that phagocytose C. albicans cells as well as the average number of C. albicans cells phagocytosed by individual S2 or RAW cells. A second line of research focused on the transcriptional regulator Wor1, a master regulator of white-opaque switching. Wor1 belongs to a family of proteins lacking similarity to any known DNA-binding protein but conserved across the fungal kingdom. My work determined that Wor1 binds DNA directly, identified the DNA sequence recognized by Wor1, and demonstrated that this sequence is sufficient for Wor1-dependent activation of transcriptional in vivo. This conserved domain, which we have termed the WOPR box, represents a distinct and previously unidentified family of DNA-binding proteins. Finally, we investigated the order of regulatory changes during the switch from the opaque to the white cell type. Surprisingly, changes in key transcriptional regulators occur gradually, extending over several cell divisions with little cell-to-cell variation. Additional experiments, including perturbations

The human fungal pathogen Candida albicans undergoes a switch between two distinct cell types, referred to as white and opaque. These cell types differ in cell and colony morphologies, ability to mate, metabolic preferences, preferred niches in the host, and interactions with the host innate immune system. Both cell types are stable through many generations; switching between them is rare, stochastic, and occurs without any known changes in the primary sequence of the genome; thus the switch is epigenetic. When I started my work, the circuit regulating this switch had just been identified and recently published results suggested that the innate immune system recognized the two cell types differently. My work determined that both the Drosophila hemocyte-derived S2 cell line and the mouse macrophage-derived RAW264.7 cell lines preferentially phagocytose white cells over opaque cells. This difference is observed for both the percentage of S2 or RAW cells that phagocytose C. albicans cells as well as the average number of C. albicans cells phagocytosed by individual S2 or RAW cells. A second line of research focused on the transcriptional regulator Wor1, a master regulator of white-opaque switching. Wor1 belongs to a family of proteins lacking similarity to any known DNA-binding protein but conserved across the fungal kingdom. My work determined that Wor1 binds DNA directly, identified the DNA sequence recognized by Wor1, and demonstrated that this sequence is sufficient for Wor1-dependent activation of transcriptional in vivo. This conserved domain, which we have termed the WOPR box, represents a distinct and previously unidentified family of DNA-binding proteins. Finally, we investigated the order of regulatory changes during the switch from the opaque to the white cell type. Surprisingly, changes in key transcriptional regulators occur gradually, extending over several cell divisions with little cell-to-cell variation. Additional experiments, including perturbations

The human fungal pathogen Candida albicans undergoes a switch between two distinct cell types, referred to as white and opaque. These cell types differ in cell and colony morphologies, ability to mate, metabolic preferences, preferred niches in the host, and interactions with the host innate immune system. Both cell types are stable through many generations; switching between them is rare, stochastic, and occurs without any known changes in the primary sequence of the genome; thus the switch is epigenetic. When I started my work, the circuit regulating this switch had just been identified and recently published results suggested that the innate immune system recognized the two cell types differently. My work determined that both the Drosophila hemocyte-derived S2 cell line and the mouse macrophage-derived RAW264.7 cell lines preferentially phagocytose white cells over opaque cells. This difference is observed for both the percentage of S2 or RAW cells that phagocytose C. albicans cells as well as the average number of C. albicans cells phagocytosed by individual S2 or RAW cells. A second line of research focused on the transcriptional regulator Wor1, a master regulator of white-opaque switching. Wor1 belongs to a family of proteins lacking similarity to any known DNA-binding protein but conserved across the fungal kingdom. My work determined that Wor1 binds DNA directly, identified the DNA sequence recognized by Wor1, and demonstrated that this sequence is sufficient for Wor1-dependent activation of transcriptional in vivo. This conserved domain, which we have termed the WOPR box, represents a distinct and previously unidentified family of DNA-binding proteins. Finally, we investigated the order of regulatory changes during the switch from the opaque to the white cell type. Surprisingly, changes in key transcriptional regulators occur gradually, extending over several cell divisions with little cell-to-cell variation. Additional experiments, including perturbations

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.

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.