scholarly journals Transcription Factor ADS-4 Regulates Adaptive Responses and Resistance to Antifungal Azole Stress

2015 ◽  
Vol 59 (9) ◽  
pp. 5396-5404 ◽  
Author(s):  
Kangji Wang ◽  
Zhenying Zhang ◽  
Xi Chen ◽  
Xianyun Sun ◽  
Cheng Jin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Efflux Pump ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Bzip Transcription Factor ◽  
Azole Resistance ◽  
Homologous Gene ◽  
Adaptive Responses ◽  
Transcriptional Responses ◽  
Content Type

ABSTRACTAzoles are commonly used as antifungal drugs or pesticides to control fungal infections in medicine and agriculture. Fungi adapt to azole stress by rapidly activating the transcription of a number of genes, and transcriptional increases in some azole-responsive genes can elevate azole resistance. The regulatory mechanisms that control transcriptional responses to azole stress in filamentous fungi are not well understood. This study identified a bZIP transcription factor, ADS-4 (antifungaldrugsensitive-4), as a new regulator of adaptive responses and resistance to antifungal azoles. Transcription ofads-4inNeurospora crassacells increased when they were subjected to ketoconazole treatment, whereas the deletion ofads-4resulted in hypersensitivity to ketoconazole and fluconazole. In contrast, the overexpression ofads-4increased resistance to fluconazole and ketoconazole inN. crassa. Transcriptome sequencing (RNA-seq) analysis, followed by quantitative reverse transcription (qRT)-PCR confirmation, showed that ADS-4 positively regulated the transcriptional responses of at least six genes to ketoconazole stress inN. crassa. The gene products of four ADS-4-regulated genes are known contributors to azole resistance, including the major efflux pump CDR4 (Pdr5p ortholog), an ABC multidrug transporter (NcAbcB), sterol C-22 desaturase (ERG5), and a lipid transporter (NcRTA2) that is involved in calcineurin-mediated azole resistance. Deletion of theads-4-homologous gene Afads-4inAspergillus fumigatuscaused hypersensitivity to itraconazole and ketoconazole, which suggested that ADS-4 is a functionally conserved regulator of adaptive responses to azoles. This study provides important information on a new azole resistance factor that could be targeted by a new range of antifungal pesticides and drugs.

scholarly journals The C2H2 Transcription Factor SltA Contributes to Azole Resistance by Coregulating the Expression of the Drug Target Erg11A and the Drug Efflux Pump Mdr1 in Aspergillus fumigatus

2021 ◽  
Author(s):  
Wenlong Du ◽  
Pengfei Zhai ◽  
Tingli Wang ◽  
Michael J Bromley ◽  
Yuanwei Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogens ◽  
Efflux Pump ◽  
Antifungal Drugs ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Efflux ◽  
Mobility Shift ◽  
Drug Efflux Pump

The emergence of azole-resistant fungal pathogens has posed a great threat to public health worldwide. Although the molecular mechanism of azole resistance has been extensively investigated, the potential regulators of azole resistance remain largely unexplored. Here we identified a new function of the fungal specific C2H2 zinc finger transcription factor SltA (involved in salt-tolerance pathway) in the regulation of azole resistance of the human fungal pathogen Aspergillus fumigatus. Lack of SltA results in an itraconazole hypersusceptibility phenotype. Transcriptional profiling combined with LacZ reporter analysis and electrophoretic mobility shift assays (EMSA) demonstrate that SltA is involved in its own transcriptional regulation and also regulates the expression of genes related to ergosterol biosynthesis (erg11A, erg13A and erg24A) and drug efflux pumps (mdr1, mfsC and abcE) by directly binding to the conserved 5’-AGGCA-3’ motif in their promoter regions, and this binding is dependent on the conserved cysteine and histidine within the C2H2 DNA binding domain of SltA. Moreover, overexpression of erg11A or mdr1 rescues sltA deletion defects under itraconazole conditions, suggesting that erg11A and mdr1 are related to sltA-mediated itraconazole resistance. Most importantly, deletion of SltA in laboratory-derived and clinical azole-resistant isolates significantly attenuates drug resistance. Collectively, we have identified a new function of the transcription factor SltA in regulating azole resistance by coordinately mediating the key azole target Erg11A and the drug efflux pump Mdr1, and targeting SltA may provide a potential strategy for intervention of clinical azole-resistant isolates to improve the efficiency of currently approved antifungal drugs.

scholarly journals Transcription Factor CCG-8 as a New Regulator in the Adaptation to Antifungal Azole Stress

2013 ◽  
Vol 58 (3) ◽  
pp. 1434-1442 ◽  
Author(s):  
Xianyun Sun ◽  
Kangji Wang ◽  
Xinxu Yu ◽  
Jie Liu ◽  
Hanxing Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Filamentous Fungi ◽  
Fungal Infections ◽  
Fusarium Verticillioides ◽  
Transporter Gene ◽  
Phospholipid Synthesis ◽  
Wild Type ◽  
Sensitivity Testing ◽  
Transcriptional Responses ◽  
Content Type

ABSTRACTAntifungal azoles are widely used for controlling fungal infections. Fungi are able to change the expression of many genes when they adapt to azole stress, and increased expression of some of these genes can elevate resistance to azoles. However, the regulatory mechanisms behind transcriptional adaption to azoles in filamentous fungi are poorly understood. In this study, we found that deletion of the transcription factor geneccg-8, which is known to be a clock-controlled gene, madeNeurospora crassahypersensitive to azoles. A comparative genome-wide analysis of the responses to ketoconazole of the wild type and theccg-8mutant revealed that the transcriptional responses to ketoconazole of 78 of the 488 transcriptionally ketoconazole-upregulated genes and the 427 transcriptionally ketoconazole-downregulated genes in the wild type were regulated by CCG-8. Ketoconazole sensitivity testing of all available knockout mutants for CCG-8-regulated genes revealed that CCG-8 contributed to azole adaption by regulating the ketoconazole responses of many genes, including the target gene (erg11), an azole transporter gene (cdr4), a hexose transporter gene (hxt13), a stress response gene (locus number NCU06317, namedkts-1), two transcription factor genes (NCU01386 [namedkts-2] andfsd-1/ndt80), four enzyme-encoding genes, and six unknown-function genes. CCG-8 also regulated phospholipid synthesis inN. crassain a manner similar to that of its homolog inSaccharomyces cerevisiae, Opi1p. However, there was no cross talk between phospholipid synthesis and azole resistance inN. crassa. CCG-8 homologs are conserved and are common in filamentous fungi. Deletion of the CCG-8 homolog-encoding gene inFusarium verticillioides(Fvccg-8) also made this fungus hypersensitive to antifungal azoles.

scholarly journals A Systematic Screen Reveals a Diverse Collection of Medications That Induce Antifungal Resistance inCandidaSpecies

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Arielle Butts ◽  
Parker Reitler ◽  
Andrew T. Nishimoto ◽  
Christian DeJarnette ◽  
Leanna R. Estredge ◽  
...  
Keyword(s):  
Treatment Failure ◽  
Amphotericin B ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Azole Resistance ◽  
Surface Mode ◽  
Drug Efflux ◽  
Content Type ◽  
Zinc Cluster ◽  
Antagonistic Interactions

ABSTRACTThe increasing incidence of and high mortality rates associated with invasive fungal infections (IFIs) impose an enormous clinical, social, and economic burden on humankind. In addition to microbiological resistance to existing antifungal drugs, the large number of unexplained treatment failures is a serious concern. Due to the extremely limited therapeutic options available, it is critical to identify and understand the various causes of treatment failure if patient outcomes are to improve. In this study, we examined one potential source of treatment failure: antagonistic drug interactions. Using a simple screen, we systematically identified currently approved medications that undermine the antifungal activity of three major antifungal drugs—fluconazole, caspofungin, and amphotericin B—on four prevalent human fungal pathogens—Candida albicans,Candida glabrata,Candida parapsilosis, andCandida tropicalis. This revealed that a diverse collection of structurally distinct drugs exhibit antagonistic interactions with fluconazole. Several antagonistic agents selected for follow-up studies induce azole resistance through a mechanism that depends on Tac1p/Pdr1p zinc-cluster transcription factors, which activate the expression of drug efflux pumps belonging to the ABC-type transporter family. Few antagonistic interactions were identified with caspofungin or amphotericin B, possibly reflecting their cell surface mode of action that should not be affected by drug efflux mechanisms. Given that patients at greatest risk of IFIs usually receive a multitude of drugs to treat various underlying conditions, these studies suggest that chemically inducible azole resistance may be much more common and important than previously realized.

scholarly journals Efflux-Mediated Antifungal Drug Resistance

2009 ◽  
Vol 22 (2) ◽  
pp. 291-321 ◽  
Author(s):  
Richard D. Cannon ◽  
Erwin Lamping ◽  
Ann R. Holmes ◽  
Kyoko Niimi ◽  
Philippe V. Baret ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Efflux Pump ◽  
Fungal Infections ◽  
Efflux Pumps ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Model Systems ◽  
Attributable Mortality ◽  
Azole Resistance ◽  
Antifungal Drug Resistance

SUMMARY Fungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.

scholarly journals Fungal Zn(II)2Cys6 Transcription Factor ADS-1 Regulates Drug Efflux and Ergosterol Metabolism Under Antifungal Azole Stress

2020 ◽  
Author(s):  
Yajing Yin ◽  
Hanxing Zhang ◽  
Yu Zhang ◽  
Chengcheng Hu ◽  
Xianyun Sun ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Efflux Pump ◽  
Transcriptional Response ◽  
Fungal Species ◽  
Antifungal Drugs ◽  
Regulatory Function ◽  
Ergosterol Biosynthesis ◽  
Agricultural Practice ◽  
Transcriptional Responses ◽  
Basal Resistance

Antifungal azoles are the most widely used antifungal drugs in clinical and agricultural practice. Fungi can make adaptive responses to azole stress by modifying transcript levels of many genes and the responsive mechanisms to azoles are the basis for fungi to develop azole resistance. In this study, we identified a new Zn(II)2Cys6 transcription factor ADS-1 with a positive regulatory function in transcriptional responses to azole stress in the model filamentous fungal species Neurospora crassa. Under ketoconazole (KTC) stress, the transcript level of ads-1 was significantly increased in N. crassa. Deletion of ads-1 increased the susceptibility to different azoles while its overexpression increased resistance to these azoles. The gene cdr4, which encodes the key azole efflux pump, was positively regulated by ADS-1. Deletion of ads-1 reduced the transcriptional response by cdr4 to KTC stress and increased the cellular KTC accumulation under KTC stress while its overexpression had the opposite effect. ADS-1 also positively regulated transcriptional response by erg11, which encodes the azole target lanosterol 14α-demethylase for ergosterol biosynthesis, to KTC stress. After KTC treatment, the ads-1 deletion mutant had less ergosterol but accumulated more lanosterol than wild type, while ads-1 overexpression had the opposite effects. The homologs of ADS-1 widely present in filamentous fungal species of Ascomycota but not in yeasts. Deletion of the gene encoding ADS-1 homolog in Aspergillus flavus also increased the susceptibility to KTC and itraconazole (ITZ). Besides, deletion of Afads-1 significantly reduced the transcriptional response by genes encoding homologs of CDR4 and ERG11 in A. flavus to KTC stress and accumulated more KTC but less ergosterol. Together, the function and regulatory mechanism of ADS-1 homologs among different fungal species in azole responses and the basal resistance of azoles are highly conserved.

scholarly journals Reversal of Azole Resistance inCandida albicansby Sulfa Antibacterial Drugs

2017 ◽  
Vol 62 (3) ◽  
Author(s):  
Hassan E. Eldesouky ◽  
Abdelrahman Mayhoub ◽  
Tony R. Hazbun ◽  
Mohamed N. Seleem
Keyword(s):  
Treatment Options ◽  
Antifungal Drugs ◽  
Infection Model ◽  
Azole Resistance ◽  
Global Public Health ◽  
Sulfa Drugs ◽  
Antibacterial Drugs ◽  
Content Type

ABSTRACTInvasive candidiasis presents an emerging global public health challenge due to the emergence of resistance to the frontline treatment options, such as fluconazole. Hence, the identification of other compounds capable of pairing with fluconazole and averting azole resistance would potentially prolong the clinical utility of this important group. In an effort to repurpose drugs in the field of antifungal drug discovery, we explored sulfa antibacterial drugs for the purpose of reversing azole resistance inCandida. In this study, we assembled and investigated a library of 21 sulfa antibacterial drugs for their ability to restore fluconazole sensitivity inCandida albicans. Surprisingly, the majority of assayed sulfa drugs (15 of 21) were found to exhibit synergistic relationships with fluconazole by checkerboard assay with fractional inhibitory concentration index (ΣFIC) values ranging from <0.0312 to 0.25. Remarkably, five sulfa drugs were able to reverse azole resistance in a clinically achievable range. The structure-activity relationships (SARs) of the amino benzene sulfonamide scaffold as antifungal agents were studied. We also identified the possible mechanism of the synergistic interaction of sulfa antibacterial drugs with azole antifungal drugs. Furthermore, the ability of sulfa antibacterial drugs to inhibitCandidabiofilm by 40%in vitrowas confirmed. In addition, the effects of sulfa-fluconazole combinations onCandidagrowth kinetics and efflux machinery were explored. Finally, using aCaenorhabditis elegansinfection model, we demonstrated that the sulfa-fluconazole combination does possess potent antifungal activityin vivo, reducingCandidain infected worms by ∼50% compared to the control.

scholarly journals Magnitude of Voriconazole Resistance in Clinical and Environmental Isolates of Aspergillus flavus and Investigation into the Role of Multidrug Efflux Pumps

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Raees A. Paul ◽  
Shivaprakash M. Rudramurthy ◽  
Manpreet Dhaliwal ◽  
Pankaj Singh ◽  
Anup K. Ghosh ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Azole Resistance ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Environmental Isolates ◽  
Content Type ◽  
Multidrug Efflux Pumps

ABSTRACT The magnitude of azole resistance in Aspergillus flavus and its underlying mechanism is obscure. We evaluated the frequency of azole resistance in a collection of clinical (n = 121) and environmental isolates (n = 68) of A. flavus by the broth microdilution method. Six (5%) clinical isolates displayed voriconazole MIC greater than the epidemiological cutoff value. Two of these isolates with non-wild-type MIC were isolated from same patient and were genetically distinct, which was confirmed by amplified fragment length polymorphism analysis. Mutations associated with azole resistance were not present in the lanosterol 14-α demethylase coding genes (cyp51A, cyp51B, and cyp51C). Basal and voriconazole-induced expression of cyp51A homologs and various efflux pump genes was analyzed in three each of non-wild-type and wild-type isolates. All of the efflux pump genes screened showed low basal expression irrespective of the azole susceptibility of the isolate. However, the non-wild-type isolates demonstrated heterogeneous overexpression of many efflux pumps and the target enzyme coding genes in response to induction with voriconazole (1 μg/ml). The most distinctive observation was approximately 8- to 9-fold voriconazole-induced overexpression of an ortholog of the Candida albicans ATP binding cassette (ABC) multidrug efflux transporter, Cdr1, in two non-wild-type isolates compared to those in the reference strain A. flavus ATCC 204304 and other wild-type strains. Although the dominant marker of azole resistance in A. flavus is still elusive, the current study proposes the possible role of multidrug efflux pumps, especially that of Cdr1B overexpression, in contributing azole resistance in A. flavus.

scholarly journals Aspergillus fumigatusAfssn3-Afssn8Pair Reverse Regulates Azole Resistance by Conferring Extracellular Polysaccharide, Sphingolipid Pathway Intermediates, and Efflux Pumps to Biofilm

2018 ◽  
Vol 62 (3) ◽  
Author(s):  
Nanbiao Long ◽  
Liping Zeng ◽  
Shanlei Qiao ◽  
Lei Li ◽  
Guowei Zhong
Keyword(s):  
Amino Acids ◽  
Aspergillus Fumigatus ◽  
Biofilm Formation ◽  
Efflux Pump ◽  
Extracellular Polysaccharide ◽  
Azole Resistance ◽  
Drug Penetration ◽  
Reverse Genetic ◽  
Content Type ◽  
Barrier Layers

ABSTRACTAntifungal treatment is often ineffectual, partly because of biofilm formation. In this study, by using a combined forward and reverse genetic strategy, we identified that nucleus-localized AfSsn3 and its partner AfSsn8, which constitute a Cdk8-cyclin pair, are required for azole resistance inAspergillus fumigatus. Deletion ofAfssn3led to increased absorption and utilization of glucose and amino acids. Interestingly, absorption and utilization of glucose accelerated the extracellular polysaccharide formation, while utilization of the amino acids serine, threonine, and glycine increased sphingolipid pathway intermediate accumulation. In addition, the absence ofAfssn3induced the activity of the efflux pump proteins. These factors indicate the mature biofilm is responsible for the major mechanisms ofA. fumigatusresistance to azoles in the ΔAfssn3mutant. Collectively, the loss ofAfssn3led to two “barrier” layers between the intracellular and extracellular spaces, which consequently decreased drug penetration into the cell.

scholarly journals Acylhydrazones as Antifungal Agents Targeting the Synthesis of Fungal Sphingolipids

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Cristina Lazzarini ◽  
Krupanandan Haranahalli ◽  
Robert Rieger ◽  
Hari Krishna Ananthula ◽  
Pankaj B. Desai ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antifungal Agents ◽  
Fungal Infections ◽  
Antifungal Drugs ◽  
Fungal Resistance ◽  
Content Type ◽  
Highly Active ◽  
Pharmacokinetic Properties ◽  
Pass Through

ABSTRACTThe incidence of invasive fungal infections has risen dramatically in recent decades. Current antifungal drugs are either toxic, likely to interact with other drugs, have a narrow spectrum of activity, or induce fungal resistance. Hence, there is a great need for new antifungals, possibly with novel mechanisms of action. Previously our group reported an acylhydrazone called BHBM that targeted the sphingolipid pathway and showed strong antifungal activity against several fungi. In this study, we screened 19 derivatives of BHBM. Three out of 19 derivatives were highly active againstCryptococcus neoformansin vitroand had low toxicity in mammalian cells. In particular, one of them, called D13, had a high selectivity index and showed better activity in an animal model of cryptococcosis, candidiasis, and pulmonary aspergillosis. D13 also displayed suitable pharmacokinetic properties and was able to pass through the blood-brain barrier. These results suggest that acylhydrazones are promising molecules for the research and development of new antifungal agents.

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.

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