RESUMEN
Protein kinases are central components of almost all signaling pathways that control cellular activities. In the model organism Saccharomyces cerevisiae, the paralogous protein kinases Ypk1 and Ypk2, which control membrane lipid homeostasis, are essential for viability, and previous studies strongly indicated that this is also the case for their single ortholog Ypk1 in the pathogenic yeast Candida albicans. Here, using FLP-mediated inducible gene deletion, we reveal that C. albicans ypk1Δ mutants are viable but slow-growing, explaining prior failures to obtain null mutants. Phenotypic analyses of the mutants showed that the functions of Ypk1 in regulating sphingolipid biosynthesis and cell membrane lipid asymmetry are conserved, but the consequences of YPK1 deletion are milder than in S. cerevisiae. Mutational studies demonstrated that the highly conserved PDK1 phosphorylation site T548 in its activation loop is essential for Ypk1 function, whereas the TORC2 phosphorylation sites S687 and T705 at the C-terminus are important for Ypk1-dependent resistance to membrane stress. Unexpectedly, Pkh1, the single C. albicans orthologue of Pkh1/Pkh2, which mediate Ypk1 phosphorylation at the PDK1 site in S. cerevisiae, was not required for normal growth of C. albicans under nonstressed conditions, and Ypk1 phosphorylation at T548 was only slightly reduced in pkh1Δ mutants. We found that another protein kinase, Pkh3, whose ortholog in S. cerevisiae cannot substitute Pkh1/2, acts redundantly with Pkh1 to activate Ypk1 in C. albicans. No phenotypic effects were observed in cells lacking Pkh3 alone, but pkh1Δ pkh3Δ double mutants had a severe growth defect and Ypk1 phosphorylation at T548 was completely abolished. These results establish that Ypk1 is not essential for viability in C. albicans and that, despite its generally conserved function, the Ypk1 signaling pathway is rewired in this pathogenic yeast and includes a novel upstream kinase to activate Ypk1 by phosphorylation at the PDK1 site.
Asunto(s)
Proteínas Quinasas , Proteínas de Saccharomyces cerevisiae , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/genética , FosforilaciónRESUMEN
The signals that denote mammalian host environments and dictate the activation of signaling pathways in human-associated microorganisms are often unknown. The transcription regulator Rtg1/3 in the human fungal pathogen Candida albicans is a crucial determinant of host colonization and pathogenicity. Rtg1/3's activity is controlled, in part, by shuttling the regulator between the cytoplasm and nucleus of the fungus. The host signal(s) that Rtg1/3 respond(s) to, however, have remained unclear. Here we report that neutrophil-derived reactive oxygen species (ROS) direct the subcellular localization of this C. albicans transcription regulator. Upon engulfment of Candida cells by human or mouse neutrophils, the regulator shuttles to the fungal nucleus. Using genetic and chemical approaches to disrupt the neutrophils' oxidative burst, we establish that the oxidants produced by the NOX2 complex-but not the oxidants generated by myeloperoxidase-trigger Rtg1/3's migration to the nucleus. Furthermore, screening a collection of C. albicans kinase deletion mutants, we implicate the MKC1 signaling pathway in the ROS-dependent regulation of Rtg1/3 in this fungus. Finally, we show that Rtg1/3 contributes to C. albicans virulence in the nematode Caenorhabditis elegans in an ROS-dependent manner as the rtg1 and rtg3 mutants display virulence defects in wild-type but not in ROS deficient worms. Our findings establish NOX2-derived ROS as a key signal that directs the activity of the pleiotropic fungal regulator Rtg1/3.
Asunto(s)
Candida albicans , Neutrófilos , Animales , Ratones , Humanos , Especies Reactivas de Oxígeno/metabolismo , Neutrófilos/metabolismo , Candida , Oxidantes/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , MamíferosRESUMEN
Protein kinases play central roles in virtually all signaling pathways that enable organisms to adapt to their environment. Microbial pathogens must cope with severely restricted iron availability in mammalian hosts to invade and establish themselves within infected tissues. To uncover protein kinase signaling pathways that are involved in the adaptation of the pathogenic yeast Candida albicans to iron limitation, we generated a comprehensive protein kinase deletion mutant library of a wild-type strain. Screening of this library revealed that the protein kinase Ire1, which has a conserved role in the response of eukaryotic cells to endoplasmic reticulum stress, is essential for growth of C. albicans under iron-limiting conditions. Ire1 was not necessary for the activity of the transcription factor Sef1, which regulates the response of the fungus to iron limitation, and Sef1 target genes that are induced by iron depletion were normally upregulated in ire1Δ mutants. Instead, Ire1 was required for proper localization of the high-affinity iron permease Ftr1 to the cell membrane. Intriguingly, iron limitation did not cause increased endoplasmic reticulum stress, and the transcription factor Hac1, which is activated by Ire1-mediated removal of the non-canonical intron in the HAC1 mRNA, was dispensable for Ftr1 localization to the cell membrane and growth under iron-limiting conditions. Nevertheless, expression of a pre-spliced HAC1 copy in ire1Δ mutants restored Ftr1 localization and rescued the growth defects of the mutants. Both ire1Δ and hac1Δ mutants were avirulent in a mouse model of systemic candidiasis, indicating that an appropriate response to endoplasmic reticulum stress is important for the virulence of C. albicans. However, the specific requirement of Ire1 for the functionality of the high-affinity iron permease Ftr1, a well-established virulence factor, even in the absence of endoplasmic reticulum stress uncovers a novel Hac1-independent essential role of Ire1 in iron acquisition and virulence of C. albicans.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Candida albicans/metabolismo , Candidiasis/microbiología , Hierro/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Candida albicans/genética , Candida albicans/patogenicidad , ADN de Hongos , Estrés del Retículo Endoplásmico , Femenino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Ratones Endogámicos BALB C , Proteínas Serina-Treonina Quinasas/genética , Eliminación de Secuencia , Transducción de Señal , Organismos Libres de Patógenos Específicos , VirulenciaRESUMEN
Increasing resistance against antimycotic drugs challenges anti-infective therapies today and contributes to the mortality of infections by drug-resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'-dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole-susceptible and fluconazole-resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.
Asunto(s)
Candida albicans , Fluconazol , Candida auris , Relación Estructura-Actividad , Antifúngicos/química , Pruebas de Sensibilidad MicrobianaRESUMEN
The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C. albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C. albicans czf1Δ mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C. albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall.
Asunto(s)
Candida albicans/metabolismo , Pared Celular/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Antifúngicos/farmacología , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Caspofungina/farmacología , Pared Celular/inmunología , Pared Celular/metabolismo , Eliminación de Gen , Neutrófilos/inmunología , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/fisiologíaRESUMEN
Candida parapsilosis is a common cause of invasive candidiasis worldwide and is the most commonly is7olated Candida species among pediatric and neonatal populations. Previous work has demonstrated that nonsynonymous mutations in the gene encoding the putative transcription factor CpMrr1 can influence fluconazole susceptibility. However, the direct contribution of these mutations and how they influence fluconazole resistance in clinical isolates are poorly understood. We identified 7 nonsynonymous CpMRR1 mutations in 12 isolates from within a collection of 35 fluconazole-resistant clinical isolates. The mutations leading to the A854V, R479K, and I283R substitutions were further examined and found to be activating mutations leading to increased fluconazole resistance. In addition to CpMDR1, we identified two other genes, one encoding a major facilitator superfamily (MFS) transporter (CpMDR1B, CPAR2_603010) and one encoding an ATP-binding cassette (ABC) transporter (CpCDR1B, CPAR2_304370), as being upregulated in isolates carrying CpMRR1-activating mutations. Overexpression of CpMDR1 in a susceptible strain and disruption in resistant clinical isolates that overexpress CpMDR1 had little to no effect on fluconazole susceptibility. Conversely, overexpression of either CpMDR1B or CpCDR1B increased resistance, and disruption in clinical isolates overexpressing these genes decreased fluconazole resistance. Our findings suggest that activating mutations in CpMRR1 represent important genetic determinants of fluconazole resistance in clinical isolates of C. parapsilosis, and unlike what is observed in Candida albicans, this is primarily driven by upregulation of both MFS (CpMdr1B) and ABC (CpCdr1B) transporters.
Asunto(s)
Candida parapsilosis , Farmacorresistencia Fúngica , Fluconazol , Transportadoras de Casetes de Unión a ATP/genética , Antifúngicos/farmacología , Candida albicans/genética , Candida albicans/metabolismo , Candida parapsilosis/efectos de los fármacos , Candida parapsilosis/metabolismo , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Humanos , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
Mutations in genes encoding zinc cluster transcription factors (ZCFs) such as TAC1, MRR1, and UPC2 play a key role in Candida albicans azole antifungal resistance. Artificial activation of the ZCF Mrr2 has shown increased expression of the gene encoding the Cdr1 efflux pump and resistance to fluconazole. Amino acid substitutions in Mrr2 have recently been reported to contribute to fluconazole resistance in clinical isolates. In the present study, 57 C. albicans clinical isolates with elevated fluconazole MICs were examined for mutations in MRR2 and expression of CDR1 Mutations in MRR2 resulting in 15 amino acid substitutions were uniquely identified among resistant isolates, including 4 substitutions (S466L, A468G, S469T, T470N) previously reported to reduce fluconazole susceptibility. Three additional, novel amino acid substitutions (R45Q, A459T, V486M) were also discovered in fluconazole-resistant isolates. When introduced into a fluconazole-susceptible background, no change in fluconazole MIC or CDR1 expression was observed for any of the mutations found in this collection. However, introduction of an allele leading to artificial activation of Mrr2 increased resistance to fluconazole as well as CDR1 expression. Moreover, Mrr2 amino acid changes reported previously to have the strongest effect on fluconazole susceptibility and CDR1 expression also exhibited no differences in fluconazole susceptibility or CDR1 expression relative to the parent strain. While all known fluconazole resistance mechanisms are represented within this collection of clinical isolates and contribute to fluconazole resistance to different extents, mutations in MRR2 do not appear to alter CDR1 expression or contribute to resistance in any of these isolates.
Asunto(s)
Candida albicans/efectos de los fármacos , Candida albicans/metabolismo , Fluconazol/farmacología , Proteínas Fúngicas/metabolismo , Factores de Transcripción/metabolismo , Azoles/farmacología , Candida albicans/genética , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Pruebas de Sensibilidad Microbiana , Mutación/genética , Factores de Transcripción/genéticaRESUMEN
The fungal Cyp51-specific inhibitors VT-1161 and VT-1598 have emerged as promising new therapies to combat fungal infections, including Candida spp. To evaluate their in vitro activities compared to other azoles, MICs were determined by Clinical and Laboratory Standards Institute (CLSI) method for VT-1161, VT-1598, fluconazole, voriconazole, itraconazole, and posaconazole against 68 C. albicans clinical isolates well characterized for azole resistance mechanisms and mutant strains representing individual azole resistance mechanisms. VT-1161 and VT-1598 demonstrated potent activity (geometric mean MICs ≤0.15 µg/ml) against predominantly fluconazole-resistant (≥8 µg/ml) isolates. However, five of 68 isolates exhibited MICs greater than six dilutions (>2 µg/ml) to both tetrazoles compared to fluconazole-susceptible isolates. Four of these isolates likewise exhibited high MICs beyond the upper limit of the assay for all triazoles tested. A premature stop codon in ERG3 likely explained the high-level resistance in one isolate. VT-1598 was effective against strains with hyperactive Tac1, Mrr1, and Upc2 transcription factors and against most ERG11 mutant strains. VT-1161 MICs were elevated compared to the control strain SC5314 for hyperactive Tac1 strains and two strains with Erg11 substitutions (Y132F and Y132F&K143R) but showed activity against hyperactive Mrr1 and Upc2 strains. While mutations affecting Erg3 activity appear to greatly reduce susceptibility to VT-1161 and VT-1598, the elevated MICs of both tetrazoles for four isolates could not be explained by known azole resistance mechanisms, suggesting the presence of undescribed resistance mechanisms to triazole- and tetrazole-based sterol demethylase inhibitors.
Asunto(s)
Antifúngicos/farmacología , Azoles/farmacología , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Piridinas/farmacología , Tetrazoles/farmacología , Candida albicans/genética , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Proteínas Fúngicas/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación/genética , Factores de Transcripción/genéticaRESUMEN
The opportunistic fungal pathogen Candida albicans frequently produces genetically altered variants to adapt to environmental changes and new host niches in the course of its life-long association with the human host. Gain-of-function mutations in zinc cluster transcription factors, which result in the constitutive upregulation of their target genes, are a common cause of acquired resistance to the widely used antifungal drug fluconazole, especially during long-term therapy of oropharyngeal candidiasis. In this study, we investigated if C. albicans also can develop resistance to the antimicrobial peptide histatin 5, which is secreted in the saliva of humans to protect the oral mucosa from pathogenic microbes. As histatin 5 has been shown to be transported out of C. albicans cells by the Flu1 efflux pump, we screened a library of C. albicans strains that contain artificially activated forms of all zinc cluster transcription factors of this fungus for increased FLU1 expression. We found that a hyperactive Mrr1, which confers fluconazole resistance by upregulating the multidrug efflux pump MDR1 and other genes, also causes FLU1 overexpression. Similarly to the artificially activated Mrr1, naturally occurring gain-of-function mutations in this transcription factor also caused FLU1 upregulation and increased histatin 5 resistance. Surprisingly, however, Mrr1-mediated histatin 5 resistance was mainly caused by the upregulation of MDR1 instead of FLU1, revealing a previously unrecognized function of the Mdr1 efflux pump. Fluconazole-resistant clinical C. albicans isolates with different Mrr1 gain-of-function mutations were less efficiently killed by histatin 5, and this phenotype was reverted when MRR1 was deleted. Therefore, antimycotic therapy can promote the evolution of strains that, as a consequence of drug resistance mutations, simultaneously have acquired increased resistance against an innate host defense mechanism and are thereby better adapted to certain host niches.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Candida albicans/genética , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Humanos , Mutación/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genéticaRESUMEN
The commensal species Candida parapsilosis is an emerging human pathogen that has the ability to form biofilms. In this study, we explored the impact of the divalent cations cobalt (Co2+), copper (Cu2+), iron (Fe3+), manganese (Mn2+), nickel (Ni2+) and zinc (Zn2+) on biofilm formation of clinical isolates of C. parapsilosis with no, low and high biofilm forming abilities at 30 and 37°C. All strains besides one isolate showed a concentration-dependent enhancement of biofilm formation at 30°C in the presence of Mn2+ with a maximum at 2 mM. The biofilm forming ability of no and low biofilm forming isolates was >2-fold enhanced in the presence of 2 mM Mn2+, while the effect in high biofilm forming isolate was significantly less pronounced. Of note, cells in the biofilms of no and low biofilm forming strains differentiated into yeast and pseudohyphal cells similar in morphology to high biofilm formers. The biofilm transcriptional activator BCR1 has a dual developmental role in the absence and presence of 2 mM Mn2+ as it promoted biofilm formation of no biofilm forming strains, and, surprisingly, suppressed cells of no biofilm forming strains to develop into pseudohyphae and/or hyphae. Thus, environmental conditions can significantly affect the amount of biofilm formation and cell morphology of C. parapsilosis with Mn2+ to overcome developmental blocks to trigger biofilm formation and to partially relieve BCR1 suppressed cell differentiation.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida parapsilosis/efectos de los fármacos , Candidiasis/microbiología , Cationes Bivalentes/farmacología , Proteínas Fúngicas/metabolismo , Manganeso/farmacología , Biopelículas/efectos de los fármacos , Candida parapsilosis/citología , Candida parapsilosis/crecimiento & desarrollo , Diferenciación Celular/efectos de los fármacos , Proteínas Fúngicas/genética , Humanos , Hifa/citología , Hifa/efectos de los fármacos , Hifa/crecimiento & desarrollo , Eliminación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The metabolic flexibility of the opportunistic fungal pathogen Candida albicans is important for colonisation and infection of different host niches. Complex regulatory networks, in which protein kinases play central roles, link metabolism and other virulence-associated traits, such as filamentous growth and stress resistance, and thereby control commensalism and pathogenicity. By screening a protein kinase deletion mutant library that was generated in the present work using an improved SAT1 flipper cassette, we found that the previously uncharacterised kinase Sak1 is a key upstream activator of the protein kinase Snf1, a highly conserved regulator of nutrient stress responses that is essential for viability in C. albicans. The sak1Δ mutants failed to grow on many alternative carbon sources and were hypersensitive to cell wall/membrane stress. These phenotypes were mirrored in mutants lacking other subunits of the SNF1 complex and partially compensated by a hyperactive form of Snf1. Transcriptional profiling of sak1Δ mutants showed that Sak1 ensures basal expression of glyoxylate cycle and gluconeogenesis genes even in glucose-rich media and thereby contributes to the metabolic plasticity of C. albicans. In a mouse model of gastrointestinal colonisation, sak1Δ mutants were rapidly outcompeted by wild-type cells, demonstrating that Sak1 is essential for the in vivo fitness of C. albicans.
Asunto(s)
Candida albicans/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Candida albicans/genética , Carbono/metabolismo , Femenino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Biblioteca de Genes , Ratones , Ratones Endogámicos BALB C , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , VirulenciaRESUMEN
Gain-of-function mutations in the zinc cluster transcription factors Mrr1, Tac1, and Upc2, which result in constitutive overexpression of their target genes, are a frequent cause of fluconazole resistance in the pathogenic yeast Candida albicans In this study, we show that an activated form of another zinc cluster transcription factor, Stb5, confers resistance to the natural compound beauvericin via the overexpression of YOR1, encoding an efflux pump of the ATP-binding cassette transporter superfamily. Beauvericin was recently shown to potentiate the activity of azole drugs against C. albicans Although Yor1 did not contribute to fluconazole resistance when C. albicans cells were treated with the drug alone, Stb5-mediated YOR1 overexpression diminished the synergistic effect of the fluconazole-beauvericin combination, thereby enhancing fluconazole resistance in beauvericin-treated C. albicans cells. Stb5-mediated YOR1 overexpression also suppressed the inhibition of hyphal growth, an important virulence trait of C. albicans, by beauvericin. Therefore, activating mutations in Stb5, which result in constitutive YOR1 overexpression, may enable C. albicans to acquire resistance to beauvericin and thereby overcome both the sensitization to azole drugs and the inhibition of morphogenesis caused by this compound.
Asunto(s)
Candida albicans/efectos de los fármacos , Candida albicans/genética , Depsipéptidos/farmacología , Proteínas Fúngicas/metabolismo , Antifúngicos/farmacología , Candida albicans/aislamiento & purificación , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Humanos , Oligomicinas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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 and Tac1, which result in constitutive overexpression of multidrug efflux pumps, and Upc2, which result in constitutive overexpression of ergosterol biosynthesis genes. However, the deregulated gene expression that is caused by hyperactive forms of these transcription factors also reduces the fitness of the cells in the absence of the drug. To investigate whether fluconazole-resistant clinical C. albicans isolates have overcome the fitness costs of drug resistance, we assessed the relative fitness of C. albicans isolates containing resistance mutations in these transcription factors in competition with matched drug-susceptible isolates from the same patients. Most of the fluconazole-resistant isolates were outcompeted by the corresponding drug-susceptible isolates when grown in rich medium without fluconazole. On the other hand, some resistant isolates with gain-of-function mutations in MRR1 did not exhibit reduced fitness under these conditions. In a mouse model of disseminated candidiasis, three out of four tested fluconazole-resistant clinical isolates did not exhibit a significant fitness defect. However, all four fluconazole-resistant isolates were outcompeted by the matched susceptible isolates in a mouse model of gastrointestinal colonization, demonstrating that the effects of drug resistance on in vivo fitness depend on the host niche. Collectively, our results indicate that the fitness costs of drug resistance in C. albicans are not easily remediated, especially when proper control of gene expression is required for successful adaptation to life within a mammalian host.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Fluconazol/farmacología , Candida albicans/genética , Candida albicans/metabolismo , Farmacorresistencia Fúngica/genética , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Mutación/genéticaRESUMEN
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.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Factores de Transcripción/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Sitios de Unión , Farmacorresistencia Fúngica/efectos de los fármacos , Fluconazol/farmacología , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Zinc/metabolismoRESUMEN
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.
Asunto(s)
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Factores de Transcripción/metabolismo , Candida albicans/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Estructura Terciaria de Proteína , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Hifa/genética , Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Candida/efectos de los fármacos , Candida/aislamiento & purificación , Candida/patogenicidad , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candida albicans/aislamiento & purificación , Candida albicans/patogenicidad , Candidiasis/microbiología , Eliminación de Gen , Prueba de Complementación Genética , Humanos , Hifa/efectos de los fármacos , Hifa/patogenicidad , Pruebas de Sensibilidad Microbiana , Factores de Virulencia/genéticaRESUMEN
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.
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Candida albicans/genética , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Zinc/metabolismo , Antifúngicos/metabolismo , Candida albicans/citología , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica , Fluconazol/metabolismo , Genética Microbiana/métodos , Biología Molecular/métodos , Micología/métodosRESUMEN
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.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Candida albicans/genética , Proteínas de Ciclo Celular/genética , Farmacorresistencia Fúngica Múltiple/genética , Proteínas Fúngicas/genética , Regulación hacia Arriba/genética , Candida albicans/efectos de los fármacos , Fluconazol/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/genética , Peróxido de Hidrógeno/farmacología , Mutación/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Micro-organisms 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 grown in the presence of low or 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 mep1Δ mep2Δ 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.
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Compuestos de Amonio/metabolismo , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Péptido Hidrolasas/metabolismo , Arginina/metabolismo , Candida albicans/enzimología , Candida albicans/genética , Proteínas Fúngicas/genética , Péptido Hidrolasas/genética , Transporte de ProteínasRESUMEN
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.