scholarly journals Candida glabrata Drug:H+Antiporter CgQdr2 Confers Imidazole Drug Resistance, Being Activated by Transcription Factor CgPdr1

2013 ◽  
Vol 57 (7) ◽  
pp. 3159-3167 ◽  
Author(s):  
Catarina Costa ◽  
Carla Pires ◽  
Tânia R. Cabrito ◽  
Adeline Renaudin ◽  
Michiyo Ohno ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Transcription Factor ◽  
Multidrug Resistance ◽  
Candida Glabrata ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Major Facilitator Superfamily ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Antifungal Drug Resistance

ABSTRACTThe widespread emergence of antifungal drug resistance poses a severe clinical problem. Though predicted to play a role in this phenomenon, the drug:H+antiporters (DHA) of the major facilitator superfamily have largely escaped characterization in pathogenic yeasts. This work describes the first DHA from the pathogenic yeastCandida glabratareported to be involved in antifungal drug resistance, theC. glabrata QDR2(CgQDR2) gene (ORFCAGL0G08624g). The expression ofCgQDR2inC. glabratawas found to confer resistance to the antifungal drugs miconazole, tioconazole, clotrimazole, and ketoconazole. By use of a green fluorescent protein (GFP) fusion, the CgQdr2 protein was found to be targeted to the plasma membrane inC. glabrata. In agreement with these observations,CgQDR2expression was found to decrease the intracellular accumulation of radiolabeled clotrimazole inC. glabrataand to play a role in the extrusion of this antifungal from preloaded cells. Interestingly, the functional heterologous expression ofCgQDR2in the model yeastSaccharomyces cerevisiaefurther confirmed the role of this gene as a multidrug resistance determinant: its expression was able to complement the susceptibility phenotype exhibited by itsS. cerevisiaehomologue,QDR2, in the presence of imidazoles and of the antimalarial and antiarrhythmic drug quinidine. In contrast to the findings reported for Qdr2, CgQdr2 expression does not contribute to the ability of yeast to grow under K+-limiting conditions. Interestingly,CgQDR2transcript levels were seen to be upregulated inC. glabratacells challenged with clotrimazole or quinidine. This upregulation was found to depend directly on the transcription factor CgPdr1, the major regulator of multidrug resistance in this pathogenic yeast, which has also been found to be a determinant of quinidine and clotrimazole resistance inC. glabrata.

scholarly journals Dysfunction of Prohibitin 2 Results in Reduced Susceptibility to Multiple Antifungal Drugs via Activation of the Oxidative Stress-Responsive Transcription Factor Pap1 in Fission Yeast

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Qiannan Liu ◽  
Fan Yao ◽  
Guanglie Jiang ◽  
Min Xu ◽  
Si Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Drug Resistance ◽  
Transcription Factor ◽  
Fission Yeast ◽  
Mitochondrial Protein ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Gene Encoding ◽  
Content Type ◽  
Antifungal Drug Resistance

ABSTRACT The fight against resistance to antifungal drugs requires a better understanding of the underlying cellular mechanisms. In order to gain insight into the mechanisms leading to antifungal drug resistance, we performed a genetic screen on a model organism, Schizosaccharomyces pombe, to identify genes whose overexpression caused resistance to antifungal drugs, including clotrimazole and terbinafine. We identified the phb2+ gene, encoding a highly conserved mitochondrial protein, prohibitin (Phb2), as a novel determinant of reduced susceptibility to multiple antifungal drugs. Unexpectedly, deletion of the phb2+ gene also exhibited antifungal drug resistance. Overexpression of the phb2+ gene failed to cause drug resistance when the pap1+ gene, encoding an oxidative stress-responsive transcription factor, was deleted. Furthermore, pap1+ mRNA expression was significantly increased when the phb2+ gene was overexpressed or deleted. Importantly, either overexpression or deletion of the phb2+ gene stimulated the synthesis of NO and reactive oxygen species (ROS), as measured by the cell-permeant fluorescent NO probe DAF-FM DA (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate) and the ROS probe DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate), respectively. Taken together, these results suggest that Phb2 dysfunction results in reduced susceptibility to multiple antifungal drugs by increasing NO and ROS synthesis due to dysfunctional mitochondria, thereby activating the transcription factor Pap1 in fission yeast.

scholarly journals The Two-Component Response Regulator Ssk1 and the Mitogen-Activated Protein Kinase Hog1 Control Antifungal Drug Resistance and Cell Wall Architecture of Candida auris

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Raju Shivarathri ◽  
Sabrina Jenull ◽  
Anton Stoiber ◽  
Manju Chauhan ◽  
Rounik Mazumdar ◽  
...  
Keyword(s):  
Cell Wall ◽  
Multidrug Resistance ◽  
Response Regulator ◽  
Multidrug Resistant ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Candida Auris ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
Two Component

ABSTRACT Candida auris is an emerging multidrug-resistant human fungal pathogen refractory to treatment by several classes of antifungal drugs. Unlike other Candida species, C. auris can adhere to human skin for prolonged periods of time, allowing for efficient skin-to-skin transmission in the hospital environments. However, molecular mechanisms underlying pronounced multidrug resistance and adhesion traits are poorly understood. Two-component signal transduction and mitogen-activated protein (MAP) kinase signaling are important regulators of adherence, antifungal drug resistance, and virulence. Here, we report that genetic removal of SSK1 encoding a response regulator and the mitogen-associated protein kinase HOG1 restores the susceptibility to both amphotericin B (AMB) and caspofungin (CAS) in C. auris clinical strains. The loss of SSK1 and HOG1 alters membrane lipid permeability, cell wall mannan content, and hyperresistance to cell wall-perturbing agents. Interestingly, our data reveal variable functions of SSK1 and HOG1 in different C. auris clinical isolates, suggesting a pronounced genetic plasticity affecting cell wall function, stress adaptation, and multidrug resistance. Taken together, our data suggest that targeting two-component signal transduction systems could be suitable for restoring C. auris susceptibility to antifungal drugs. IMPORTANCE Candida auris is an emerging multidrug-resistant (MDR) fungal pathogen that presents a serious global threat to human health. The Centers for Disease Control and Prevention (CDC) have classified C. auris as an urgent threat to public health for the next decade due to its major clinical and economic impact and the lack of effective antifungal drugs and because of future projections concerning new C. auris infections. Importantly, the Global Antimicrobial Resistance Surveillance System (GLASS) has highlighted the need for more robust and efficacious global surveillance schemes enabling the identification and monitoring of antifungal resistance in Candida infections. Despite the clinical relevance of C. auris infections, our overall understanding of its pathophysiology and virulence, its response to human immune surveillance, and the molecular basis of multiple antifungal resistance remains in its infancy. Here, we show a marked phenotypic plasticity of C. auris clinical isolates. Further, we demonstrate critical roles of stress response mechanisms in regulating multidrug resistance and show that cell wall architecture and composition are key elements that determine antifungal drug susceptibilities. Our data promise new therapeutic options to treat drug-refractory C. auris infections.

scholarly journals Environmental Isolates of Azole-Resistant Aspergillus fumigatus in Germany

2015 ◽  
Vol 59 (7) ◽  
pp. 4356-4359 ◽  
Author(s):  
Oliver Bader ◽  
Jana Tünnermann ◽  
Anna Dudakova ◽  
Marut Tangwattanachuleeporn ◽  
Michael Weig ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Aspergillus Fumigatus ◽  
Antifungal Drug ◽  
Clinical Samples ◽  
Environmental Isolates ◽  
Northern Germany ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
The World

ABSTRACTAzole antifungal drug resistance inAspergillus fumigatusis an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR34/L98H and TR46/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.

scholarly journals Effects of Azole Antifungal Drugs on the Transition from Yeast Cells to Hyphae in Susceptible and Resistant Isolates of the Pathogenic Yeast Candida albicans

1999 ◽  
Vol 43 (4) ◽  
pp. 763-768 ◽  
Author(s):  
Kien C. Ha ◽  
Theodore C. White
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Molecular Mechanisms ◽  
Opportunistic Infections ◽  
Antifungal Drugs ◽  
Yeast Cells ◽  
Pathogenic Yeast ◽  
Oral Infections ◽  
Antifungal Drug Resistance ◽  
Hyphal Formation

ABSTRACT Oral infections caused by the yeast Candida albicansare some of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug resistance, creating a major problem in the treatment of yeast infections in AIDS patients and other immunocompromised individuals. Several molecular mechanisms that contribute to drug resistance have been identified. InC. albicans, the ability to morphologically switch from yeast cells (blastospores) to filamentous forms (hyphae) is an important virulence factor which contributes to the dissemination ofCandida in host tissues and which promotes infection and invasion. A positive correlation between the level of antifungal drug resistance and the ability to form hyphae in the presence of azole drugs has been identified. Under hypha-inducing conditions in the presence of an azole drug, resistant clinical isolates form hyphae, while susceptible yeast isolates do not. This correlation is observed in a random sample from a population of susceptible and resistant isolates and is independent of the mechanisms of resistance.35S-methionine incorporation suggests that growth inhibition is not sufficient to explain the inhibition of hyphal formation, but it may contribute to this inhibition.

scholarly journals Functional Dissection of a Candida albicans Zinc Cluster Transcription Factor, the Multidrug Resistance Regulator Mrr1

2011 ◽  
Vol 10 (8) ◽  
pp. 1110-1121 ◽  
Author(s):  
Sabrina Schubert ◽  
Christina Popp ◽  
P. David Rogers ◽  
Joachim Morschhäuser
Keyword(s):  
Transcription Factor ◽  
Candida Albicans ◽  
Transcriptional Activation ◽  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Constitutive Activity ◽  
Activation Domain ◽  
Pathogenic Yeast ◽  
Content Type ◽  
Zinc Cluster

ABSTRACTThe overexpression of theMDR1gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the widely used antimycotic agent fluconazole and other toxic compounds in the pathogenic yeastCandida albicans. The zinc cluster transcription factor Mrr1 controlsMDR1expression in response to inducing chemicals, and gain-of-function mutations inMRR1are responsible for the constitutiveMDR1upregulation in fluconazole-resistantC. albicansstrains. To understand how Mrr1 activity is regulated, we identified functional domains of this transcription factor. A hybrid protein consisting of the N-terminal 106 amino acids of Mrr1 and the transcriptional activation domain of Gal4 fromSaccharomyces cerevisiaeconstitutively inducedMDR1expression, demonstrating that the DNA binding domain is sufficient to target Mrr1 to theMDR1promoter. Using a series of C-terminal truncations and systematic internal deletions, we could show that Mrr1 contains multiple activation and inhibitory domains. One activation domain (AD1) is located in the C terminus of Mrr1. When fused to the tetracycline repressor TetR, this distal activation domain induced gene expression from a TetR-dependent promoter. The deletion of an inhibitory region (ID1) located near the distal activation domain resulted in constitutive activity of Mrr1. The additional removal of AD1 abolished the constitutive activity, but the truncated Mrr1 still could activate theMDR1promoter in response to the inducer benomyl. These results demonstrate that the activity of Mrr1 is regulated in multiple ways and provide insights into the function of an important mediator of drug resistance inC. albicans.

scholarly journals A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Qusai Al Abdallah ◽  
Wenbo Ge ◽  
Jarrod R. Fortwendel
Keyword(s):  
Drug Resistance ◽  
Gene Deletion ◽  
Gene Replacement ◽  
Antifungal Drug ◽  
Wild Type ◽  
Gene Manipulation ◽  
Content Type ◽  
Antifungal Drug Resistance ◽  
Efficient Gene

ABSTRACT Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in Aspergillus fumigatus. However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA. To overcome such limitations, we developed a simple and universal CRISPR-Cas9 system for gene deletion that works across different genetic backgrounds of A. fumigatus. The system employs in vitro assembly of dual Cas9 ribonucleoproteins (RNPs) for targeted gene deletion. Additionally, our CRISPR-Cas9 system utilizes 35 to 50 bp of flanking regions for mediating homologous recombination at Cas9 double-strand breaks (DSBs). As a proof of concept, we first tested our system in the ΔakuB (ΔakuB ku80 ) laboratory strain and generated high rates (97%) of gene deletion using 2 µg of the repair template flanked by homology regions as short as 35 bp. Next, we inspected the portability of our system across other genetic backgrounds of A. fumigatus, namely, the wild-type strain Af293 and a clinical isolate, A. fumigatus DI15-102. In the Af293 strain, 2 µg of the repair template flanked by 35 and 50 bp of homology resulted in highly efficient gene deletion (46% and 74%, respectively) in comparison to classical gene replacement systems. Similar deletion efficiencies were also obtained in the clinical isolate DI15-102. Taken together, our data show that in vitro-assembled Cas9 RNPs coupled with microhomology repair templates are an efficient and universal system for gene manipulation in A. fumigatus. IMPORTANCE Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus.

scholarly journals The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009582
Author(s):  
Bao Gia Vu ◽  
Mark A. Stamnes ◽  
Yu Li ◽  
P. David Rogers ◽  
W. Scott Moye-Rowley
Keyword(s):  
Transcription Factor ◽  
Membrane Protein ◽  
Candida Glabrata ◽  
Target Genes ◽  
Protein Biosynthesis ◽  
Antifungal Drugs ◽  
Azole Resistance ◽  
Mutant Form ◽  
Pathogenic Yeast ◽  
Genes Encoding

The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

scholarly journals Multiple mechanisms impact fluconazole resistance of mutant Erg11 proteins in Candida glabrata

2021 ◽  
Author(s):  
Bao Vu ◽  
W. Scott Moye-Rowley
Keyword(s):  
Transcription Factor ◽  
Candida Glabrata ◽  
Double Mutant ◽  
Antifungal Drugs ◽  
Cross Resistance ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Pathogenic Yeast ◽  
Fluconazole Resistance ◽  
Growth Defects

Azoles remain the most common used antifungal drugs for invasive candidiasis worldwide. They specifically inhibit the fungal lanosterol a-14 demethylase enzyme, which is commonly referred to as Erg11 in fungi. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased azole resistance. Candida glabrata is also a pathogenic yeast that has a low intrinsic susceptibility to azole drugs and easily develops elevated resistance. These azole resistant mutations are almost exclusively found to cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance associated mutations in C. albicans ERG11. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. The azole resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. These data support the view that C. glabrata does not typically acquire ERG11 mutations owing to growth defects associated with these lesions while hyperactive PDR1 alleles have no obvious growth issues. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.

scholarly journals Examining Signatures of Natural Selection in Antifungal Resistance Genes Across Aspergillus Fungi

2021 ◽  
Vol 2 ◽  
Author(s):  
Renato Augusto Corrêa dos Santos ◽  
Matthew E. Mead ◽  
Jacob L. Steenwyk ◽  
Olga Rivero-Menéndez ◽  
Ana Alastruey-Izquierdo ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Natural Selection ◽  
Positive Selection ◽  
Resistance Genes ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Sequence Evolution ◽  
Antifungal Drug Resistance ◽  
Selective Pressures ◽  
Substantial Heterogeneity

Certain Aspergillus fungi cause aspergillosis, a set of diseases that typically affect immunocompromised individuals. Most cases of aspergillosis are caused by Aspergillus fumigatus, which infects millions of people annually. Some closely related so-called cryptic species, such as Aspergillus lentulus, can also cause aspergillosis, albeit at lower frequencies, and they are also clinically relevant. Few antifungal drugs are currently available for treating aspergillosis and there is increasing worldwide concern about the presence of antifungal drug resistance in Aspergillus species. Furthermore, isolates from both A. fumigatus and other Aspergillus pathogens exhibit substantial heterogeneity in their antifungal drug resistance profiles. To gain insights into the evolution of antifungal drug resistance genes in Aspergillus, we investigated signatures of positive selection in 41 genes known to be involved in drug resistance across 42 susceptible and resistant isolates from 12 Aspergillus section Fumigati species. Using codon-based site models of sequence evolution, we identified ten genes that contain 43 sites with signatures of ancient positive selection across our set of species. None of the sites that have experienced positive selection overlap with sites previously reported to be involved in drug resistance. These results identify sites that likely experienced ancient positive selection in Aspergillus genes involved in resistance to antifungal drugs and suggest that historical selective pressures on these genes likely differ from any current selective pressures imposed by antifungal drugs.

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