[Show abstract][Hide abstract] ABSTRACT: The pathogenic yeast Candida albicans can develop resistance to azole antifungal drugs by overexpressing ERG11, which encodes the drug target, or the multidrug efflux pumps MDR1 and CDR1/CDR2. The constitutive upregulation of these genes is usually caused by gain-of-function mutations in the zinc cluster transcription
factors Upc2, Mrr1, and Tac1, respectively. These transcription factors are also required for the induction of their target
genes in drug-susceptible strains in the presence of specific stimuli. By swapping the DNA-binding domains of Mrr1, Tac1,
and Upc2 we investigated if the hybrid transcription factors could activate their new target genes in response to the same
signals. When Tac1 was targeted to the MDR1 and ERG11 promoters, the expression of these genes became inducible by fluphenazine. Similarly, MDR1 and CDR2 were strongly upregulated by fluconazole when Upc2 was fused to the DNA-binding domains of Mrr1 and Tac1, respectively. In
contrast, Mrr1 was unable to promote gene expression in response to benomyl when it was targeted to the CDR2 and ERG11 promoters instead of the MDR1 promoter. These results suggest that Tac1 and Upc2 themselves are activated by the inducers fluphenazine and fluconazole,
respectively, whereas benomyl does not activate Mrr1 itself but a coregulatory factor that is present at the promoters of
Mrr1 target genes. Strains in which the expression levels of Mrr1 and Tac1 target genes were controlled by Upc2 exhibited
increased fluconazole resistance, demonstrating that the ability to efficiently upregulate the expression of efflux pumps
in the presence of the drug results in enhanced intrinsic fluconazole resistance.
Preview · Article · Nov 2014 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Overexpression of the multidrug efflux pump MDR1 is one mechanism by which the pathogenic yeast Candida albicans develops resistance to the antifungal drug fluconazole. The constitutive upregulation of MDR1 in fluconazole-resistant, clinical C. albicans isolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that
Mrr1 activates MDR1 transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However, MDR1 expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response in C. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotes MDR1 overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 caused MDR1 overexpression in a wild-type strain but only weakly in mutants lacking ADA2. In the presence of benomyl or H2O2, compounds that induce MDR1 expression in an Mrr1- and Cap1-dependent fashion, MDR1 was upregulated with the same efficiency in wild-type and ada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to the MDR1 promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required for MDR1 overexpression in fluconazole-resistant C. albicans strains containing gain-of-function mutations in Mrr1.
Full-text · Article · Jun 2014 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Microorganisms sense the availability of nutrients in their environment to control cellular behaviour and the expression of transporters and enzymes that are required for the utilization of these nutrients. In the pathogenic yeast Candida albicans, the preferred nitrogen source ammonium suppresses the switch from yeast to filamentous growth in response to certain stimuli and it also represses the secretion of proteases, which are required for the utilization of proteins as an alternative nitrogen source. To investigate whether C. albicans senses the availability of ammonium in the extracellular environment or if ammonium uptake into the cell is required to regulate morphogenesis and gene expression, we compared the behaviour of wild-type cells and ammonium uptake-deficient mutants in the presence and absence of extracellular ammonium. Arginine-induced filamentous growth was suppressed by ammonium in the wild type, but not in mutants lacking the ammonium permeases Mep1 and Mep2. Similarly, ammonium suppressed protease secretion and extracellular protein degradation in the wild type, but not in mutants lacking the ammonium transporters. By comparing the gene expression profiles of C. albicans in the presence of low and high ammonium concentrations, we identified a set of genes whose expression is controlled by nitrogen availability. The repression of genes involved in the utilization of alternative nitrogen sources, which occurred under ammonium-replete conditions in the wild type, was abrogated in mep1Capital Greek Delta mep2Capital Greek Delta mutants. These results demonstrate that C. albicans does not respond to the presence of sufficient amounts of the preferred nitrogen source ammonium by sensing its availability in the environment. Instead, ammonium has to be taken up into the cell to control morphogenesis, protease secretion and gene expression.
[Show abstract][Hide abstract] ABSTRACT: In Candida parapsilosis, biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates
causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative,
low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which
strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance
to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis, has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature
biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and
RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation
in clinical isolates of C. parapsilosis is both dependent and independent of BCR1, but even in strains which showed a BCR1-independent biofilm phenotype, BCR1 has alternative physiological functions.
[Show abstract][Hide abstract] ABSTRACT: Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white) to an elongated cell type (opaque), which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11(ΔN467)) efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11(ΔN467)-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase.
[Show abstract][Hide abstract] ABSTRACT: The zinc cluster proteins are a family of transcription factors that are unique to the fungal kingdom. In the pathogenic yeast Candida albicans, zinc cluster transcription factors control the expression of virulence-associated traits and play key roles in the development of antifungal drug resistance. Gain-of-function mutations in several zinc cluster transcription factors, which result in constitutive overexpression of their target genes, are a frequent cause of azole resistance in clinical C. albicans isolates. We found that zinc cluster proteins can also be artificially activated by C-terminal fusion with the heterologous Gal4 activation domain. We used this strategy to create a comprehensive library of C. albicans strains expressing all 82 zinc cluster transcription factors of this fungus in a potentially hyperactive form. Screening of this library identified regulators of invasive filamentous growth and other phenotypes that are important during an infection. In addition, the approach uncovered several novel mediators of fluconazole resistance, including the multidrug resistance regulator Mrr2, which controls the expression of the major C. albicans multidrug efflux pump CDR1. Artificial activation therefore is a highly useful method to study the role of zinc cluster transcription factors in C. albicans and other fungi of medical, agricultural, and biotechnological importance.
No preview · Article · Jul 2013 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: Candida albicans and Candida dubliniensis are pathogenic fungi that are highly related but differ in virulence and in some phenotypic traits. During in vitro growth on certain nutrient-poor media, C. albicans and C. dubliniensis are the only yeast species which are able to produce chlamydospores, large thick-walled cells of unknown function. Interestingly, only C. dubliniensis forms pseudohyphae with abundant chlamydospores when grown on Staib medium, while C. albicans grows exclusively as a budding yeast. In order to further our understanding of chlamydospore development and assembly, we compared the global transcriptional profile of both species during growth in liquid Staib medium by RNA sequencing. We also included a C. albicans mutant in our study which lacks the morphogenetic transcriptional repressor Nrg1. This strain, which is characterized by its constitutive pseudohyphal growth, specifically produces masses of chlamydospores in Staib medium, similar to C. dubliniensis. This comparative approach identified a set of putatively chlamydospore-related genes. Two of the homologous C. albicans and C. dubliniensis genes (CSP1 and CSP2) which were most strongly upregulated during chlamydospore development were analysed in more detail. By use of the green fluorescent protein as a reporter, the encoded putative cell wall related proteins were found to exclusively localize to C. albicans and C. dubliniensis chlamydospores. Our findings uncover the first chlamydospore specific markers in Candida species and provide novel insights in the complex morphogenetic development of these important fungal pathogens.
[Show abstract][Hide abstract] ABSTRACT: The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms
of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed
that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only
the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine
tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis
identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible
SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed
mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing
agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.
[Show abstract][Hide abstract] ABSTRACT: Fungi possess two distinct proton-coupled peptide transport systems, the dipeptide/tripeptide transporters (PTR) and the oligopeptide
transporters (OPT), which enable them to utilize peptides as nutrients. In the pathogenic yeast Candida albicans, peptide transporters are encoded by gene families consisting of two PTR genes and eight OPT genes. To gain insight into the functions and importance of specific peptide transporters, we generated mutants lacking the
two dipeptide/tripeptide transporters Ptr2 and Ptr22, as well as the five major oligopeptide transporters Opt1 to Opt5. These
mutants were unable to grow in media containing peptides as the sole nitrogen source. Forced expression of individual peptide
transporters in the septuple mutants showed that Ptr2 and Ptr22 could utilize all tested dipeptides as substrates but differed
in their abilities to transport specific tripeptides. Interestingly, several oligopeptide transporters, which are thought
to transport peptides consisting of more than three amino acids, also mediated the uptake of tripeptides. Opt1 especially
turned out to be a highly flexible transporter that enabled growth on all tripeptides tested and could even utilize a dipeptide,
a function that has never been ascribed to this family of peptide transporters. Despite their inability to grow on proteins
or peptides, the opt1Δ opt2Δ opt3Δ opt4Δ opt5Δ ptr2Δ ptr22Δ septuple mutants had no in vivo fitness defect in a mouse model of gastrointestinal colonization. Therefore, the nutritional versatility of C. albicans enables it to utilize alternative nitrogen sources in this host niche, which probably contributes to its success as a commensal
and pathogen in mammalian hosts.
[Show abstract][Hide abstract] ABSTRACT: Candida albicans strains that are homozygous at the mating type locus can spontaneously and reversibly switch from the normal yeast morphology (white) to an elongated cell type (opaque), which is the mating-competent form of the fungus. White-opaque switching also influences the ability of C. albicans to colonize and proliferate in specific host niches and its susceptibility to host defense mechanisms. We used live imaging to observe the interaction of white and opaque cells with host phagocytic cells. For this purpose, we generated derivatives of the switching-competent strain WO-1 that express green fluorescent protein from a white-specific promoter and red fluorescent protein from an opaque-specific promoter or vice versa. When mixed populations of these differentially labeled white and opaque cells were incubated with human polymorphonuclear neutrophils (PMNs) on a glass slide, the neutrophils selectively phagocytosed and killed white cells, despite frequent physical interaction with opaque cells. White cells were attacked only after they started to form a germ tube, indicating that the suppression of filamentation in opaque cells saved them from recognition by the PMNs. In contrast to neutrophils, dendritic cells internalized white as well as opaque cells. However, when embedded in a collagen matrix, the PMNs also phagocytosed both white and opaque cells with similar efficiency. These results suggest that, depending on the environment, white-opaque switching enables C. albicans to escape from specific host defense mechanisms.
[Show abstract][Hide abstract] ABSTRACT: The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis. Resistance is often caused by gain-of-function mutations in the transcription factors Mrr1, Tac1 and Upc2, which result in constitutive overexpression of multidrug efflux pumps and ergosterol biosynthesis genes respectively. It is not known how the permanently changed gene expression program in resistant strains affects their fitness in the absence of drug selection pressure. We have systematically investigated the effects of activating mutations in Mrr1, Tac1 and Upc2, individually and in all possible combinations, on the degree of fluconazole resistance and on the fitness of C. albicans in an isogenic strain background. All combinations of different resistance mechanisms resulted in a stepwise increase in drug resistance, culminating in 500-fold increased fluconazole resistance in strains possessing mutations in the three transcription factors and an additional resistance mutation in the drug target enzyme Erg11. The acquisition of resistance mutations was associated with reduced fitness under non-selective conditions in vitro as well as in vivo during colonization of a mammalian host. Therefore, without compensatory mutations, the inability to appropriately regulate gene expression results in a loss of competitive fitness of drug-resistant C. albicans strains.
No preview · Article · Aug 2012 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: In Candida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway.
To date, three documented UPC2 gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase
in ERG11 expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole,
we found that 47 overexpressed ERG11 by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates,
29 contained a mutation in UPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations in UPC2, we recovered eight distinct mutations resulting in putative single amino acid substitutions: G648D, G648S, A643T, A643V,
Y642F, G304R, A646V, and W478C. Seven of these resulted in increased ERG11 expression, increased cellular ergosterol, and decreased susceptibility to fluconazole compared to the results for the wild-type
strain. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D,
G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in
oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal
resistance, but the findings do not account for ERG11 overexpression in all such isolates of C. albicans.
[Show abstract][Hide abstract] ABSTRACT: Overexpression of the multidrug efflux pump MDR1 confers resistance to the antifungal drug fluconazole on Candida albicans. It has been reported that two types of MDR1 promoters exist in C. albicans and that homozygosity for the allele with higher activity may promote fluconazole resistance. We found that the two MDR1 promoter alleles in strain SC5314 were equally well activated by inducing chemicals or hyperactive forms of the transcription
factors Mrr1 and Cap1, which control MDR1 expression. In addition, no loss of heterozygosity at the MDR1 locus was observed in MDR1-overexpressing clinical C. albicans strains that developed fluconazole resistance during therapy.
Preview · Article · May 2012 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: In addition to gene inactivation, the manipulation of gene expression is another highly useful tool for the analysis of gene function. Several regulatable promoters are available that enable researchers to shut off or turn on the expression of a target gene in Candida albicans, usually by growing the cells in inducing or repressing media. In this chapter, we describe a tetracycline-inducible gene expression system (Tet-On) that allows forced expression of endogenous or heterologous genes in C. albicans by the addition of the small-molecule inducer doxycycline in a growth medium-independent manner. The system is based on a cassette in which a gene of interest can be placed under the control of a Tet-inducible promoter in a single cloning step and integrated into the C. albicans genome with the help of a dominant selection marker. As the cassette contains all necessary components for Tet-inducible gene expression, it can be used to study the effect of forced gene expression on the phenotype of C. albicans cells in any strain without a requirement of additional genetic manipulations.
No preview · Article · Jan 2012 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Targeted gene inactivation is an important method to investigate gene function. In the diploid yeast Candida albicans, the generation of homozygous knock-out mutants requires the sequential replacement of both alleles of a gene by a selection marker. Targeted gene deletion is often performed in auxotrophic host strains, which are rendered prototrophic after the insertion of appropriate nutritional marker genes into the target locus. The SAT1-flipping strategy described in this chapter allows gene deletion in prototrophic C. albicans wild-type strains with the help of a recyclable dominant selection marker. The SAT1 flipper cassette used for this purpose consists of the caSAT1 marker, which confers resistance to the antibiotic nourseothricin, and the caFLP gene, which encodes the site-specific recombinase FLP. The addition of flanking sequences of the target gene allows specific genomic insertion of the SAT1 flipper cassette by homologous recombination and selection of nourseothricin-resistant transformants. Expression of the FLP recombinase results in subsequent excision of the cassette, which is bordered by direct repeats of the FLP recognition sequence FRT, from the genome. The homozygous mutants obtained after two rounds of insertion and recycling of the SAT1 flipper cassette differ from the wild-type parental strain only by the absence of the target gene and can be used for the inactivation of additional genes and the generation of complemented strains using the same strategy.
No preview · Article · Jan 2012 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine
the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG
pathway). The major oligopeptide transporters OPT1–OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport
to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a
Km of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote
member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be
crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in
the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote
from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence.
Full-text · Article · Dec 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Moulds are characterized by their saprophytic lifestyle that is based on osmotrophy. Among them, Aspergillus fumigatus has emerged as the leading cause of fungal infections in the presence of an underlying immunodeficiency. To assess the role of its nutritional versatility for virulence, transcriptional profiling studies in the presence of varying sources of nitrogen were carried out and revealed an extensive reprogramming of the fungal transcriptome when shifting to a proteinaceous growth substrate. Transcripts encoding metabolic activities were predominantly upregulated, as were proteinases and transport activities. To probe whether fundamental aspects of its osmotrophic lifestyle, that is, extracellular proteolysis and uptake of oligopeptides, are required for A. fumigatus pathogenicity, serial gene replacements were carried out, which eventually yielded an octuple deletion mutant ablated for the opt gene family. This strain displayed no growth defect on various substrates, but supplementary reduction of extracellular proteolytic activity by additional deletion of the prtT gene revealed a synthetic phenotype on porcine lung tissue agar. Virulence studies in a murine model of pulmonary aspergillosis did not disclose any attenuation in virulence of these deletants. Our data emphasize a high degree of redundancy encoded by the A. fumigatus genome that secures nutrient supply for growth and, therefore, virulence.
Full-text · Article · Nov 2011 · Molecular Microbiology
[Show abstract][Hide abstract] ABSTRACT: The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80Δ mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80Δ mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.