scholarly journals Genetic Architecture of Hsp90-Dependent Drug Resistance

2006 ◽  
Vol 5 (12) ◽  
pp. 2184-2188 ◽  
Author(s):  
Leah E. Cowen ◽  
Anne E. Carpenter ◽  
Oranart Matangkasombut ◽  
Gerald R. Fink ◽  
Susan Lindquist
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Genetic Architecture ◽  
Opportunistic Pathogen ◽  
Azole Resistance

ABSTRACT Hsp90 potentiates the evolution of azole resistance in the model yeast Saccharomyces cerevisiae and the opportunistic pathogen Candida albicans via calcineurin. Here, we explored effectors downstream of calcineurin regulating this Hsp90-dependent trait. Using S. cerevisiae erg3 mutants as a model, we determined that both Crz1 and Hph1 modulate azole resistance.

scholarly journals CaNdt80 Is Involved in Drug Resistance in Candida albicans by Regulating CDR1

2004 ◽  
Vol 48 (12) ◽  
pp. 4505-4512 ◽  
Author(s):  
Chia-Geun Chen ◽  
Yun-Liang Yang ◽  
Hsin-I Shih ◽  
Chia-Li Su ◽  
Hsiu-Jung Lo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Candida Albicans ◽  
Type Strain ◽  
Antifungal Agents ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Galactosidase Activity ◽  
Wild Type

ABSTRACT Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the β-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.

scholarly journals Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans

2000 ◽  
Vol 44 (11) ◽  
pp. 2985-2990 ◽  
Author(s):  
Hiroshi Kakeya ◽  
Yoshitsugu Miyazaki ◽  
Haruko Miyazaki ◽  
Katherine Nyswaner ◽  
Brian Grimberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Candida Albicans ◽  
Amino Acid ◽  
Genetic Analysis ◽  
Expression System ◽  
Single Copy ◽  
Gene Replacement ◽  
Azole Resistance ◽  
High Level

ABSTRACT High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

scholarly journals Put3 Positively Regulates Proline Utilization in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Walters Aji Tebung ◽  
Raha Parvizi Omran ◽  
Debra L. Fulton ◽  
Joachim Morschhäuser ◽  
Malcolm Whiteway
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Nitrogen Source ◽  
Opportunistic Pathogen ◽  
Baker's Yeast ◽  
Null Mutant ◽  
Multiple Sources ◽  
Content Type ◽  
Proline Catabolism

ABSTRACT Candida albicans poses a significant threat to the lives of immunocompromised people. Historically, knowledge has been drawn from studies on Saccharomyces cerevisiae to understand the pathogen, and many Candida albicans genes are named after their S. cerevisiae orthologs. Direct studies on the pathogen have, however, revealed differences in the roles of some orthologous proteins in the two yeasts. We show that the Put3 transcription factor allows the pathogen to completely degrade proline to usable nitrogen and carbon by evading regulatory restrictions imposed on its S. cerevisiae ortholog, which mandates conditional use of proline only as a nitrogen source in the baker’s yeast. The ability of Candida albicans to freely obtain nutrients from multiple sources may help it thrive as a commensal and opportunistic pathogen. The zinc cluster transcription factor Put3 was initially characterized in Saccharomyces cerevisiae as the transcriptional activator of PUT1 and PUT2, two genes acting early in the proline assimilation pathway. We have used phenotypic studies, transcription profiling, and chromatin immunoprecipitation with microarray technology (ChIP-chip) to establish that unlike S. cerevisiae, which only uses proline as a nitrogen source, Candida albicans can use proline as a nitrogen source, a carbon source, or a source of both nitrogen and carbon. However, a C. albicans put3 null mutant cannot grow on proline, suggesting that as in S. cerevisiae, C. albicans Put3 (CaPut3) is required for proline catabolism, and because the C. albicans put3 null mutant grew efficiently on glutamate as the sole carbon or nitrogen source, it appears that CaPut3 also regulates the early genes of the pathway. CaPut3 showed direct binding to the CaPUT1 promoter, and both PUT1 and PUT2 were upregulated in response to proline addition in a Put3-dependent manner, as well as in a C. albicans strain expressing a hyperactive Put3. CaPut3 directs proline degradation even in the presence of a good nitrogen source such as ammonia, which contrasts with S. cerevisiae Put3 (ScPut3)-regulated proline catabolism, which only occurs in the absence of a rich nitrogen source. Thus, while overall proline regulatory circuitry differs between S. cerevisiae and C. albicans, the specific role of Put3 appears fundamentally conserved. IMPORTANCE Candida albicans poses a significant threat to the lives of immunocompromised people. Historically, knowledge has been drawn from studies on Saccharomyces cerevisiae to understand the pathogen, and many Candida albicans genes are named after their S. cerevisiae orthologs. Direct studies on the pathogen have, however, revealed differences in the roles of some orthologous proteins in the two yeasts. We show that the Put3 transcription factor allows the pathogen to completely degrade proline to usable nitrogen and carbon by evading regulatory restrictions imposed on its S. cerevisiae ortholog, which mandates conditional use of proline only as a nitrogen source in the baker’s yeast. The ability of Candida albicans to freely obtain nutrients from multiple sources may help it thrive as a commensal and opportunistic pathogen.

scholarly journals Snf7p, a Component of the ESCRT-III Protein Complex, Is an Upstream Member of the RIM101 Pathway in Candida albicans

2004 ◽  
Vol 3 (6) ◽  
pp. 1609-1618 ◽  
Author(s):  
Amy L. Kullas ◽  
Mingchun Li ◽  
Dana A. Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Protein Complex ◽  
Opportunistic Pathogen ◽  
Multivesicular Bodies ◽  
Virulence Analysis ◽  
Alkaline Environments ◽  
Cognate Gene ◽  
Mutant Phenotypes ◽  
Two Hybrid

ABSTRACT The success of Candida albicans as an opportunistic pathogen is based in part on its ability to adapt to diverse environments. The RIM101 pathway governs adaptation to neutral-alkaline environments and is required for virulence. Analysis of a genomic two-hybrid study conducted with Saccharomyces cerevisiae revealed that components involved in multivesicular bodies (MVB) transport may interact with RIM101 pathway members. Thus, we hypothesized that these proteins may function in the RIM101 pathway in C. albicans. We identified C. albicans homologs to S. cerevisiae Snf7p, Vps4p, and Bro1p and generated mutants in the cognate gene. We found that snf7Δ/Δ mutants, but not vps4Δ/Δ nor bro1Δ/Δ mutants, had phenotypes similar to, but more severe than, those of RIM101 pathway mutants. We found that the constitutively active RIM101-405 allele partially rescued snf7Δ/Δ mutant phenotypes. The vps4Δ/Δ mutant had subtle phenotypes, but these were not rescued by the RIM101-405 allele. Further, we found that the snf7Δ/Δ, vps4Δ/Δ, and bro1Δ/Δ mutants did not efficiently localize the vital dye FM4-64 to the vacuole and that it was often accumulated in an MVB-like compartment. This phenotype was not rescued by RIM101-405 or observed in RIM101 pathway mutants. These results suggest that Snf7p may serve two functions in the cell: one as a RIM101 pathway member and one for MVB transport to the vacuole.

scholarly journals Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate

2021 ◽  
Vol 7 (11) ◽  
pp. 897
Author(s):  
Danyon O. Graham ◽  
Rajni K. Wilson ◽  
Yasmeen N. Ruma ◽  
Mikhail V. Keniya ◽  
Joel D. A. Tyndall ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Crystal Structures ◽  
Differential Susceptibility ◽  
Azole Resistance ◽  
Wild Type ◽  
Gene Encoding ◽  
High Level ◽  
Structural Insights ◽  
Level Resistance

Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs.

scholarly journals Overexpression of Erg11p by the RegulatableGAL1 Promoter Confers Fluconazole Resistance inSaccharomyces cerevisiae

1999 ◽  
Vol 43 (11) ◽  
pp. 2798-2800 ◽  
Author(s):  
Dimitrios P. Kontoyiannis ◽  
Namita Sagar ◽  
Kendal D. Hirschi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Azole Resistance ◽  
Fluconazole Resistance ◽  
Target Enzyme

ABSTRACT The contribution of the dosage of target enzyme P-450 14α-demethylase (14αDM) to fluconazole resistance in bothCandida albicans and Saccharomyces cerevisiaeremains unclear. Here, we show that overexpression ofSaccharomyces P-450 14αDM in S. cerevisiae, under the control of the regulatable promoter GAL1, results in azole resistance.

scholarly journals Activity of Isavuconazole and Other Azoles against Candida Clinical Isolates and Yeast Model Systems with Known Azole Resistance Mechanisms

2015 ◽  
Vol 60 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Dominique Sanglard ◽  
Alix T. Coste
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Abc Transporters ◽  
Resistance Mechanisms ◽  
Model Systems ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Limited Effect ◽  
Content Type ◽  
Major Facilitator

ABSTRACTIsavuconazole is a novel, broad-spectrum, antifungal azole. In order to evaluate its interactions with known azole resistance mechanisms, isavuconazole susceptibility among different yeast models and clinical isolates expressing characterized azole resistance mechanisms was tested and compared to those of fluconazole, itraconazole, posaconazole, and voriconazole.Saccharomyces cerevisiaeexpressing theCandida albicansandC. glabrataATP binding cassette (ABC) transporters (CDR1,CDR2, andCgCDR1), major facilitator (MDR1), and lanosterol 14-α-sterol-demethylase (ERG11) alleles with mutations were used. In addition, pairs ofC. albicansandC. glabratastrains from matched clinical isolates with known azole resistance mechanisms were investigated. The expression of ABC transporters increased all azole MICs, suggesting that all azoles tested were substrates of ABC transporters. The expression ofMDR1did not increase posaconazole, itraconazole, and isavuconazole MICs. Relative increases of azole MICs (from 4- to 32-fold) were observed for fluconazole, voriconazole, and isavuconazole when at least two mutations were present in the sameERG11allele. Upon MIC testing of azoles with clinicalC. albicansandC. glabrataisolates with known resistance mechanisms, the MIC90s ofC. albicansfor fluconazole, voriconazole, itraconazole, posaconazole, and isavuconazole were 128, 2, 1, 0.5, and 2 μg/ml, respectively, while inC. glabratathey were 128, 2, 4, 4, and 16 μg/ml, respectively. In conclusion, the effects of azole resistance mechanisms on isavuconazole did not differ significantly from those of other azoles. Resistance mechanisms in yeasts involving ABC transporters andERG11decreased the activity of isavuconazole, whileMDR1had limited effect.

scholarly journals mDrop-seq: Massively parallel single-cell RNA-seq of Saccharomyces cerevisiae and Candida albicans

2021 ◽  
Author(s):  
Ryan Dohn ◽  
Bingqing Xie ◽  
Rebecca Back ◽  
Alan Selewa ◽  
Heather Eckart ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Stress Response ◽  
Single Cell ◽  
High Throughput ◽  
Yeast Species ◽  
Model Organism ◽  
Opportunistic Pathogen ◽  
Mrna Levels ◽  
Rna Seq

Advances in high-throughput single-cell mRNA sequencing (scRNA-seq) have been limited till date by technical challenges like tough cell walls and low RNA quantity that prevented transcriptomic profiling of microbial species at throughput. We present microbial Drop-seq or mDrop-seq, a high-throughput scRNA-seq technique that is used on two yeast species, Saccharomyces cerevisiae, a popular model organism and Candida albicans, a common opportunistic pathogen. We benchmarked mDrop-seq for sensitivity and specificity and used it to profile 35,109 S. cerevisiae cells to detect variation in mRNA levels between them. As a proof of concept, we quantified expression differences in heat-shocked S. cerevisiae using mDrop-seq. We detected differential activation of stress response genes within a seemingly homogenous population of S. cerevisiae under heat-shock. We also applied mDrop-seq to C. albicans cells, a polymorphic and clinically relevant yeast species with thicker cell wall compared to S. cerevisiae. Single cell transcriptomes in 39,705 C. albicans cells was characterized using mDrop-seq under different conditions, including exposure to fluconazole, a common anti-fungal drug. We noted differential regulation in stress response and drug target pathways between C. albicans cells, changes in cell cycle patterns and marked increases in histone activity. These experiments are among the first high throughput single cell RNA-seq on different yeast species and demonstrate mDrop-seq as an affordable, easily implementable, and scalable technique that can quantify the variability in gene expression in different yeast species. We hope that mDrop-seq will lead to better understanding of genetic variation in yeasts in response to stimuli and find immediate applications in investigating drug resistance and infection outcome.

scholarly journals Cdc42p GTPase Regulates the Budded-to-Hyphal-Form Transition and Expression of Hypha-Specific Transcripts in Candida albicans

2004 ◽  
Vol 3 (3) ◽  
pp. 724-734 ◽  
Author(s):  
Alysia L. vandenBerg ◽  
Ashraf S. Ibrahim ◽  
John E. Edwards ◽  
Kurt A. Toenjes ◽  
Douglas I. Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endothelial Cells ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Opportunistic Pathogen ◽  
Integral Function ◽  
Morphological Transition ◽  
Strongly Correlated ◽  
Hypha Formation

ABSTRACT The yeast Candida albicans is a major opportunistic pathogen of immunocompromised individuals. It can grow in several distinct morphological states, including budded and hyphal forms, and the ability to make the dynamic transition between these forms is strongly correlated with virulence. Recent studies implicating the Cdc42p GTPase in hypha formation relied on cdc42 mutations that affected the mitotic functions of the protein, thereby precluding any substantive conclusions about the specific role of Cdc42p in the budded-to-hypha-form transition and virulence. Therefore, we took advantage of several Saccharomyces cerevisiae cdc42 mutants that separated Cdc42p's mitotic functions away from its role in filamentous growth. The homologous cdc42-S26I, cdc42-E100G, and cdc42-S158T mutations in C. albicans Cdc42p caused a dramatic defect in the budded-to-hypha-form transition in response to various hypha-inducing signals without affecting normal budded growth, strongly supporting the conclusion that Cdc42p has an integral function in orchestrating the morphological transition in C. albicans. In addition, the cdc42-S26I and cdc42-E100G mutants demonstrated a reduced ability to damage endothelial cells, a process that is strongly correlated to virulence. The three mutants also had reduced expression of several hypha-specific genes, including those under the regulation of the Efg1p transcription factor. These data indicate that Cdc42p-dependent signaling pathways regulate the budded-to-hypha-form transition and the expression of hypha-specific genes.

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