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 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 The Histoplasma capsulatum DDR48 Gene Is Required For Survival Within Macrophages, Response To Oxidative Stress, And Resistance to Antifungal Drugs

2020 ◽  
Author(s):  
Logan T. Blancett ◽  
Kauri A. Runge ◽  
Gabriella M. Reyes ◽  
Lauren A. Kennedy ◽  
Sydney C. Jackson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Drug Resistance ◽  
Stress Response ◽  
Virulence Factors ◽  
Histoplasma Capsulatum ◽  
The United States ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Antifungal Drug Resistance

AbstractHistoplasma capsulatum (Hc) is a systemic, dimorphic fungal pathogen that affects upwards of 500,000 individuals in the United States annually. Hc grows as a multicellular mold at environmental temperatures; whereas, upon inhalation into a human or other mammalian host, it transforms into a unicellular, pathogenic yeast. This manuscript is focused on characterizing the DNA damage-responsive gene HcDDR48. HcDDR48 was originally isolated via a subtractive DNA library enriched for transcripts enriched in the mold-phase of Hc growth. Upon further analysis we found that HcDDR48 is not just expressed in the mold morphotype, but both growth programs dependent upon the environment. We found that HcDDR48 is involved in oxidative stress response, antifungal drug resistance, and survival within resting and activated macrophages. Growth of ddr48Δ yeasts was severely decreased when exposed to the reactive oxygen species generator paraquat, as compared to wildtype controls. We also found that ddr48Δ yeasts were 2-times more sensitive to the antifungal drugs amphotericin b and ketoconazole. To test HcDDR48’s involvement in vivo, we infected resting and activated RAW 264.7 murine macrophages with Hc yeasts and measured yeast survival 24-hours post-infection. We observed a significant decrease in yeast recovery in the ddr48Δ strain compared to wildtype Hc levels. Herein, we demonstrate the importance of maintaining a functional copy of HcDDR48 in order for Hc yeasts to sense and respond to numerous environmental and host-associated stressors.ImportanceHistoplasma capsulatum is an intracellular pathogen of phagocytes, where it subverts immune recognition and avoids killing by the innate immune system. Macrophages provide a permissive environment for Hc replication and killing only occurs upon the onset of the T-cell driven adaptive immune response. Hc has evolved numerous virulence factors that aid in its survival against host-derived ROS and RNS in vivo. While these virulence factors have been described in past years, only a few reports describing the regulation of these genes and how this intricate system leads to fungal survival. In this study, we characterized the stress response gene DDR48 and determined it to be indispensable for Hc survival within macrophages. HcDDR48 regulates transcript levels of superoxide dismutases and catalases responsible for detoxification of ROS and contributes to antifungal drug resistance. Our studies highlight DDR48 as a potential target to control Hc infection and decrease the severity of the disease process.

scholarly journals Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen

2021 ◽  
Author(s):  
Shelby Priest ◽  
Vikas Yadav ◽  
Cullen Roth ◽  
Tim Dahlmann ◽  
Ulrich Kueck ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Nonsense Mutation ◽  
Fungal Pathogen ◽  
Genetic Locus ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Gene Encoding ◽  
Antifungal Drug Resistance ◽  
Trait Locus ◽  
Almost All

Abstract Microorganisms survive and compete within their environmental niches and avoid evolutionary stagnation by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes in the context of strong selective pressures. To explore how hypermutation arises in nature and elucidate its consequences, we employed a collection of 387 sequenced clinical and environmental isolates of Cryptococcus neoformans. This fungal pathogen is responsible for ~ 15% of annual AIDS-related deaths and is associated with high mortality rates, attributable to a dearth of antifungal drugs and increasing drug resistance. Isolates were screened for the ability to rapidly acquire antifungal drug resistance, and two robust hypermutators were identified. Insertion of the non-LTR Cnl1 retrotransposon was found to be responsible for the majority of drug-resistant isolates. Long-read whole-genome sequencing revealed both hypermutator genomes have two unique features: 1) hundreds of Cnl1 copies organized in subtelomeric arrays on both ends of almost all chromosomes, and 2) a nonsense mutation in the first exon of ZNF3, a gene encoding an RNAi component involved in silencing transposons. Quantitative trait locus mapping identified a significant genetic locus associated with hypermutation that includes the mutant znf3 allele, and CRISPR-mediated genome editing of the znf3 single-base pair nonsense mutation abolished the hypermutation phenotype and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from loss of RNAi combined with subsequent accumulation of a large genomic burden of a novel transposable element in C. neoformans.

scholarly journals Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen

2021 ◽  
Author(s):  
Shelby J Priest ◽  
Vikas Yadav ◽  
Cullen Roth ◽  
Tim Alexander Dahlmann ◽  
Ulrich Kuck ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Nonsense Mutation ◽  
Fungal Pathogen ◽  
Genetic Locus ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Gene Encoding ◽  
Antifungal Drug Resistance ◽  
Trait Locus ◽  
Almost All

Microorganisms survive and compete within their environmental niches and avoid evolutionary stagnation by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes in the context of strong selective pressures. To explore how hypermutation arises in nature and elucidate its consequences, we employed a collection of 387 sequenced clinical and environmental isolates of Cryptococcus neoformans. This fungal pathogen is responsible for ~15% of annual AIDS-related deaths and is associated with high mortality rates, attributable to a dearth of antifungal drugs and increasing drug resistance. Isolates were screened for the ability to rapidly acquire antifungal drug resistance, and two robust hypermutators were identified. Insertion of the non-LTR Cnl1 retrotransposon was found to be responsible for the majority of drug-resistant isolates. Long-read whole-genome sequencing revealed both hypermutator genomes have two unique features: 1) hundreds of Cnl1 copies organized in subtelomeric arrays on both ends of almost all chromosomes, and 2) a nonsense mutation in the first exon of ZNF3, a gene encoding an RNAi component involved in silencing transposons. Quantitative trait locus mapping identified a significant genetic locus associated with hypermutation that includes the mutant znf3 allele, and CRISPR-mediated genome editing of the znf3 single-base pair nonsense mutation abolished the hypermutation phenotype and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from loss of RNAi combined with subsequent accumulation of a large genomic burden of a novel transposable element in C. neoformans.

scholarly journals Mode of Selection and Experimental Evolution of Antifungal Drug Resistance in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1287-1298
Author(s):  
James B Anderson ◽  
Caroline Sirjusingh ◽  
Ainslie B Parsons ◽  
Charles Boone ◽  
Claire Wickens ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Pleiotropic Effect ◽  
Antifungal Drug ◽  
Recessive Mutation ◽  
High Concentration ◽  
Gene Encoding ◽  
Antifungal Drug Resistance ◽  
Degree Of Dominance ◽  
A Genome ◽  
Abc Transporter Genes

Abstract We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast. In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations. Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations. These mutations were semidominant and additive in their effect on resistance. The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2. These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole. In experiment 2, yeast populations were subjected to a single high concentration of fluconazole. Under this regimen, a single recessive mutation appeared in each of three replicate populations. In a genome-wide screen of ∼4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W). The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2. Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole. In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.

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 Pleiotropic Effects of the P5-Type ATPase SpfA on Stress Response Networks Contribute to Virulence in the Pathogenic Mold Aspergillus fumigatus

mBio ◽  
2021 ◽  
Author(s):  
José P. Guirao-Abad ◽  
Martin Weichert ◽  
Ginés Luengo-Gil ◽  
Sarah Sze Wah Wong ◽  
Vishukumar Aimanianda ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Resistance ◽  
Secretory Pathway ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Gene Encoding ◽  
Content Type ◽  
Downstream Target ◽  
P Type ◽  
Er Homeostasis

The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus .

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 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.

scholarly journals Candida antifungal drug resistance in sub-Saharan African populations: A systematic review

F1000Research ◽  
2017 ◽  
Vol 5 ◽  
pp. 2832 ◽  
Author(s):  
Charlene Wilma Joyce Africa ◽  
Pedro Miguel dos Santos Abrantes
Keyword(s):  
Drug Resistance ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Sub Saharan Africa ◽  
African Countries ◽  
Antifungal Drug Resistance ◽  
Resistance Patterns ◽  
Sub Saharan

Background:Candidainfections are responsible for increased morbidity and mortality rates in at-risk patients, especially in developing countries where there is limited access to antifungal drugs and a high burden of HIV co-infection. Objectives:This study aimed to identify antifungal drug resistance patterns within the subcontinent of Africa. Methods: A literature search was conducted on published studies that employed antifungal susceptibility testing on clinicalCandidaisolates from sub-Saharan African countries using Pubmed and Google Scholar. Results: A total of 21 studies from 8 countries constituted this review. Only studies conducted in sub-Saharan Africa and employing antifungal drug susceptibility testing were included. Regional differences inCandidaspecies prevalence and resistance patterns were identified. Discussion: The outcomes of this review highlight the need for a revision of antifungal therapy guidelines in regions most affected byCandidadrug resistance.  Better controls in antimicrobial drug distribution and the implementation of regional antimicrobial susceptibility surveillance programmes are required in order to reduce the highCandidadrug resistance levels seen to be emerging in sub-Saharan Africa.

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