scholarly journals A Gain-of-Function Mutation in the Transcription Factor Upc2p Causes Upregulation of Ergosterol Biosynthesis Genes and Increased Fluconazole Resistance in a Clinical Candida albicans Isolate

2008 ◽  
Vol 7 (7) ◽  
pp. 1180-1190 ◽  
Author(s):  
Nico Dunkel ◽  
Teresa T. Liu ◽  
Katherine S. Barker ◽  
Ramin Homayouni ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Ergosterol Biosynthesis ◽  
Gain Of Function ◽  
Zinc Cluster ◽  
Fluconazole Resistant ◽  
Lanosterol Demethylase

ABSTRACT In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate the expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Overexpression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Overexpression of ERG11 leads to the increased production of lanosterol demethylase, which contributes to azole resistance in clinical isolates of C. albicans, but the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately upregulated with ERG11 in a fluconazole-resistant clinical isolate compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single-nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug-susceptible strain resulted in constitutive upregulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole-resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to the increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans.

scholarly journals An A643T Mutation in the Transcription Factor Upc2p Causes Constitutive ERG11 Upregulation and Increased Fluconazole Resistance in Candida albicans

2009 ◽  
Vol 54 (1) ◽  
pp. 353-359 ◽  
Author(s):  
Clemens J. Heilmann ◽  
Sabrina Schneider ◽  
Katherine S. Barker ◽  
P. David Rogers ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Acquired Resistance ◽  
Amino Acid Position ◽  
Resistant Isolate ◽  
Ergosterol Biosynthesis ◽  
Common Mechanism ◽  
Gain Of Function ◽  
Fluconazole Resistance ◽  
Zinc Cluster

ABSTRACT The zinc cluster transcription factor Upc2p mediates upregulation of ergosterol biosynthesis genes in response to ergosterol depletion in the fungal pathogen Candida albicans. One mechanism of acquired resistance to the antifungal drug fluconazole, which inhibits ergosterol biosynthesis, is constitutively increased expression of the ERG11 gene encoding the drug target enzyme. A G648D mutation in Upc2p has recently been shown to cause hyperactivity of the transcription factor, resulting in overexpression of ergosterol biosynthesis genes and increased fluconazole resistance. In order to investigate if gain-of-function mutations in Upc2p are a common mechanism of ERG11 upregulation and fluconazole resistance, we sequenced the UPC2 alleles of four ERG11-overexpressing, fluconazole-resistant C. albicans isolates and matched susceptible isolates from the same patients. In three of the isolate pairs, no differences in the UPC2 alleles were found, suggesting that mechanisms other than Upc2p mutations can cause ERG11 overexpression. One resistant isolate had become homozygous for a UPC2 allele containing a G1927A substitution that caused an alanine-to-threonine exchange at amino acid position 643 of Upc2p. Replacement of one of the endogenous UPC2 alleles in a fluconazole-susceptible strain by the UPC2 A643T allele resulted in ERG11 overexpression and increased fluconazole resistance, which was further elevated when the A643T mutation was also introduced into the second UPC2 allele. These results further establish gain-of-function mutations in UPC2, which can be followed by loss of heterozygosity for the mutated allele, as a mechanism of ERG11 overexpression and increased fluconazole resistance in C. albicans, but other mechanisms of ERG11 upregulation also exist.

scholarly journals Gain-of-Function Mutations inUPC2Are a Frequent Cause ofERG11Upregulation in Azole-Resistant Clinical Isolates of Candida albicans

2012 ◽  
Vol 11 (10) ◽  
pp. 1289-1299 ◽  
Author(s):  
Stephanie A. Flowers ◽  
Katherine S. Barker ◽  
Elizabeth L. Berkow ◽  
Geoffrey Toner ◽  
Sean G. Chadwick ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Amino Acid ◽  
Clinical Isolates ◽  
Transcriptional Analysis ◽  
Single Amino Acid ◽  
Ergosterol Biosynthesis ◽  
Amino Acid Substitutions ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster

ABSTRACTInCandida albicans, Upc2 is a zinc-cluster transcription factor that targets genes, including those of the ergosterol biosynthesis pathway. To date, three documentedUPC2gain-of-function (GOF) mutations have been recovered from fluconazole-resistant clinical isolates that contribute to an increase inERG11expression and decreased fluconazole susceptibility. In a group of 63 isolates with reduced susceptibility to fluconazole, we found that 47 overexpressedERG11by at least 2-fold over the average expression levels in 3 unrelated fluconazole-susceptible strains. Of those 47 isolates, 29 contained a mutation inUPC2, whereas the remaining 18 isolates did not. Among the isolates containing mutations inUPC2, 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 increasedERG11expression, 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 theMDR1gene, and the uncharacterized ATP binding cassette transporterCDR11. These findings demonstrate that gain-of-function mutations inUPC2are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account forERG11overexpression in all such isolates ofC. albicans.

scholarly journals SAGA/ADA Complex Subunit Ada2 Is Required for Cap1- but Not Mrr1-Mediated Upregulation of the Candida albicans Multidrug Efflux PumpMDR1

2014 ◽  
Vol 58 (9) ◽  
pp. 5102-5110 ◽  
Author(s):  
Bernardo Ramírez-Zavala ◽  
Selene Mogavero ◽  
Eva Schöller ◽  
Christoph Sasse ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Gain Of Function ◽  
Content Type ◽  
Zinc Cluster ◽  
Fluconazole Resistant

ABSTRACTOverexpression of the multidrug efflux pumpMDR1is one mechanism by which the pathogenic yeastCandida albicansdevelops resistance to the antifungal drug fluconazole. The constitutive upregulation ofMDR1in fluconazole-resistant, clinicalC. albicansisolates is caused by gain-of-function mutations in the zinc cluster transcription factor Mrr1. It has been suggested that Mrr1 activatesMDR1transcription by recruiting Ada2, a subunit of the SAGA/ADA coactivator complex. However,MDR1expression is also regulated by the bZIP transcription factor Cap1, which mediates the oxidative stress response inC. albicans. Here, we show that a hyperactive Mrr1 containing a gain-of-function mutation promotesMDR1overexpression independently of Ada2. In contrast, a C-terminally truncated, hyperactive Cap1 causedMDR1overexpression in a wild-type strain but only weakly in mutants lackingADA2. In the presence of benomyl or H2O2, compounds that induceMDR1expression in an Mrr1- and Cap1-dependent fashion,MDR1was upregulated with the same efficiency in wild-type andada2Δ cells. These results indicate that Cap1, but not Mrr1, recruits Ada2 to theMDR1promoter to induce the expression of this multidrug efflux pump and that Ada2 is not required forMDR1overexpression in fluconazole-resistantC. albicansstrains containing gain-of-function mutations in Mrr1.

scholarly journals Competitive Fitness of Fluconazole-Resistant Clinical Candida albicans Strains

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Christina Popp ◽  
Irene A. I. Hampe ◽  
Tobias Hertlein ◽  
Knut Ohlsen ◽  
P. David Rogers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Transcription Factors ◽  
Mouse Model ◽  
Ergosterol Biosynthesis ◽  
Fitness Costs ◽  
Content Type ◽  
Constitutive Overexpression ◽  
Fluconazole Resistant

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 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.

scholarly journals Gain-of-Function Mutations in the Transcription Factor MRR1 Are Responsible for Overexpression of the MDR1 Efflux Pump in Fluconazole-Resistant Candida dubliniensis Strains

2008 ◽  
Vol 52 (12) ◽  
pp. 4274-4280 ◽  
Author(s):  
Sabrina Schubert ◽  
P. David Rogers ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Efflux Pump ◽  
Candida Dubliniensis ◽  
Major Facilitator Superfamily ◽  
Multidrug Efflux Pump ◽  
Nucleotide Substitutions ◽  
Gain Of Function ◽  
Zinc Cluster ◽  
Fluconazole Resistant

ABSTRACT Candida dubliniensis, a yeast that is closely related to Candida albicans, can rapidly develop resistance to the commonly used antifungal agent fluconazole in vitro and in vivo during antimycotic therapy. Fluconazole resistance in C. dubliniensis is usually caused by constitutive overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily. The zinc cluster transcription factor Mrr1p has recently been shown to control MDR1 expression in C. albicans in response to inducing stimuli, and gain-of-function mutations in the MRR1 gene result in constitutive upregulation of the MDR1 efflux pump. We identified a gene with a high degree of similarity to C. albicans MRR1 (CaMRR1) in the C. dubliniensis genome sequence. When C. dubliniensis MRR1 (CdMRR1) was expressed in C. albicans mrr1Δ mutants, it restored benomyl-inducible MDR1 expression, demonstrating that CdMRR1 is the ortholog of CaMRR1. To investigate whether MDR1 overexpression in C. dubliniensis is caused by mutations in MRR1, we sequenced the MRR1 alleles from a fluconazole-resistant, clinical C. dubliniensis isolate and a matched, fluconazole-susceptible isolate from the same patient as well as those from four in vitro-generated, fluconazole-resistant C. dubliniensis strains derived from two different C. dubliniensis isolates. We found that all five resistant strains contained single nucleotide substitutions or small in-frame deletions that resulted in amino acid changes in Mrr1p. Expression of these mutated alleles in C. albicans resulted in the constitutive activation of the MDR1 promoter and multidrug resistance. Therefore, mutations in MRR1 are the major cause of MDR1 upregulation in both C. albicans and C. dubliniensis, demonstrating that the transcription factor Mrr1p plays a central role in the development of drug resistance in these human fungal pathogens.

scholarly journals Mutations in TAC1B: a novel genetic determinant of clinical fluconazole resistance in C. auris

2020 ◽  
Author(s):  
Jeffrey M. Rybak ◽  
José F. Muñoz ◽  
Katherine S. Barker ◽  
Josie E. Parker ◽  
Brooke D. Esquivel ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Clinical Isolate ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Resistant Isolate ◽  
Genetic Determinant ◽  
Candida Auris ◽  
Fluconazole Resistance ◽  
Fluconazole Resistant

ABSTRACTCandida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, the fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro. Comparative analysis of comprehensive sterol profiles, [3H]-fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and the relative expression of C. auris ERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene, TAC1B, and a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into the fluconazole-susceptible isolate AR0387, was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in-vitro, and that mutations in TAC1B significantly contribute to clinical fluconazole resistance.IMPORTANCECandida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrate that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole, and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.

Antifungal Effects of Voriconazole-Loaded Nano-Liposome on Fluconazole-Resistant Clinical Isolates of Candida albicans, Biological Activity and ERG11, CDR1, and CDR2 Gene Expression

2021 ◽  
Vol 19 (7) ◽  
pp. 453-462
Author(s):  
Parviz Hassanpour ◽  
Hamed Hamishehkar ◽  
Behnaz Bahari Baroughi ◽  
Behzad Baradaran ◽  
Siamak Sandoghchian Shotorbani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Biological Activity ◽  
Candida Albicans ◽  
Clinical Isolates ◽  
Antifungal Effects ◽  
Fluconazole Resistant

scholarly journals An A643V Amino Acid Substitution in Upc2p Contributes to Azole Resistance in Well-Characterized Clinical Isolates ofCandida albicans

2010 ◽  
Vol 55 (2) ◽  
pp. 940-942 ◽  
Author(s):  
Samantha J. Hoot ◽  
Adam R. Smith ◽  
Ryan P. Brown ◽  
Theodore C. White
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Gain Of Function ◽  
Ofcandida Albicans

ABSTRACTTheCandida albicansUpc2p transcription factor regulatesERG11, encoding the target of azole drugs. Gain-of-function mutations that contribute to resistance were recently identified in a series of sequential clinical isolates (N. Dunkel, T. T. Liu, K. S. Barker, R. Homayouni, J. Morschhauser, and P. D. Rogers, Eukaryot. Cell 7:1180-1190, 2008). In the present study,UPC2was sequenced from a matched set of 17 isolates. An A643V substitution was present in all of the isolates in the series that overexpressedERG11. Azole susceptibility, ergosterol levels, and expression ofERGgenes were elevated in the A643V clinical isolates and in reconstructed strains.

scholarly journals Antifungal Activity of Thai Cajuput Oil and Its Effect on Efflux-Pump Gene Expression in Fluconazole-Resistant Candida albicans Clinical Isolates

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Pitchayaphong Keereedach ◽  
Karnjana Hrimpeng ◽  
Khaemaporn Boonbumrung
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Antifungal Activity ◽  
Efflux Pump ◽  
Clinical Problem ◽  
Antifungal Drugs ◽  
Clinical Isolates ◽  
Common Mechanism ◽  
Fluconazole Resistant ◽  
Resistant Strains

Candidiasis caused by the fluconazole-resistant opportunistic pathogen Candida albicans is an intractable clinical problem that threatens immunocompromised or normal individuals. The most common mechanism of fluconazole resistance in C. albicans is the failure of cells to accumulate the drug due to increased expression of the efflux proteins encoded by the CDR1, CDR2, and MDR1 genes. Because the number of current antifungal drugs is limited, it is necessary to develop new therapeutic strategies. This study aimed to evaluate the antifungal activity of Thai Cajuput oil, its synergism with fluconazole, and its effect on efflux-pump gene expression in fluconazole-resistant C. albicans clinical isolates. Thus, we first detected the efflux-pump genes in fourteen resistant strains by PCR. The frequencies of the CDR1, CDR2, and MDR1 genes were 68.75%, 62.5%, and 87.5%, respectively, and these efflux-pump genes were distributed in three distinct patterns. Subsequently, the antifungal activity of Thai Cajuput oil was assessed by broth macrodilution and its synergism with fluconazole was evaluated by the checkerboard assay. The changes in the expression levels of CDR1, CDR2, and MDR1 after treatment with Thai Cajuput oil were analyzed by qRT-PCR. The MICs and MFCs of Thai Cajuput oil ranged from 0.31 to 1.25 μl/ml and 0.63 to 1.25 μl/ml, respectively, and its activity was defined as fungicidal activity. The MICs of the combination of Thai Cajuput oil and fluconazole were much lower than the MICs of the individual drugs. Interestingly, sub-MICs of Thai Cajuput oil significantly reduced the MDR1 expression level in resistant strains P < 0.05 . Our study suggests that Thai Cajuput oil can be used to create new potential combination therapies to combat the antifungal resistance of C. albicans.

Sign in / Sign up

Export Citation Format

Share Document