The sterol C-14 reductase Erg24 is responsible for ergosterol biosynthesis and ion homeostasis in Aspergillus fumigatus

2021 ◽  
Vol 105 (3) ◽  
pp. 1253-1268
Author(s):  
Yeqi Li ◽  
Mengyao Dai ◽  
Yuanwei Zhang ◽  
Ling Lu
Keyword(s):  
Aspergillus Fumigatus ◽  
Ion Homeostasis ◽  
Ergosterol Biosynthesis

Point mutation or overexpression of A. fumigatus cyp51B, encoding lanosterol 14α-sterol demethylase, leads to triazole resistance.

2021 ◽  
Author(s):  
Mariana Handelman ◽  
Zohar Meir ◽  
Jennifer Scott ◽  
Yona Shadkchan ◽  
Wei Liu ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Point Mutation ◽  
Clinical Isolate ◽  
Recipient Strain ◽  
Antifungal Treatment ◽  
Ergosterol Biosynthesis ◽  
Common Cause ◽  
Clinical Resistance

Aspergillus fumigatus is the most common cause of invasive fungal mold infections in immunocompromised individuals. Current antifungal treatment relies heavily on the triazole antifungals which inhibit fungal Erg11/Cyp51 activity and subsequent ergosterol biosynthesis. However, resistance, due primarily to cyp51 mutation, is rapidly increasing. A. fumigatus contains two Cyp51 isoenzymes, Cyp51A and Cyp51B. Overexpression and mutation of Cyp51A is a major cause of triazole resistance in A. fumigatus . The role of Cyp51B in generating resistance is unclear. Here we show that overexpression or mutation of cyp51B results in triazole resistance. We demonstrate that introduction of a G457S Cyp51B mutation identified in a resistant clinical isolate, results in voriconazole resistance in the naïve recipient strain. Our results indicate that mutations in cyp51B resulting in clinical resistance do exist and should be monitored.

scholarly journals Electron donor cytochrome b5 is required for hyphal tip accumulation of sterol-rich plasma membrane domains and membrane fluidity in Aspergillus fumigatus

2020 ◽  
Author(s):  
Chi Zhang ◽  
Yiran Ren ◽  
Lu Gao ◽  
Huiyu Gu ◽  
Ling Lu
Keyword(s):  
Plasma Membrane ◽  
Aspergillus Fumigatus ◽  
Membrane Fluidity ◽  
Electron Donor ◽  
Cytochrome B5 ◽  
Normal Growth ◽  
Ergosterol Biosynthesis ◽  
Membrane Domains ◽  
Growth Defects ◽  
C Terminus

The electron donor cytochrome b5 (CybE/Cyb5) fuels the activity of the ergosterol biosynthesis-related P450 enzymes/P450s by providing electrons to P450s to promote ergosterol biosynthesis. Previous studies reported that lack of Aspergillus fumigatus (A. fumigatus) CybE reduces the proportion of ergosterol in total sterols and induces severe growth defects. However, the molecular characteristics of CybE and the underlying mechanism for CybE maintaining A. fumigatus growth remain poorly understood. Here, we found that CybE locates at the endoplasmic reticulum by its C-terminus with two transmembrane regions. Therefore, lack of the C-terminus of CybE is able to phenocopy a cybE deletion. Notably, cybE deletion reduced the accumulation of the sterol-rich plasma membrane domains (SRDs, the assembly platform of polarity factors/cell end markers and growth machinery) in hyphal tips and decreased membrane fluidity, which correspond to tardiness of hyphal extension and hypersensitivity to low temperature in cybE deletion mutant. Additionally, overexpressing another electron donor-heme-independent P450 reductase (CPR) significantly rescued growth defects and recovered SRD accumulation in deletion of cybE almost to the wild-type level, suggesting CybE maintaining the growth and deposition of SRDs in hyphal tips attributes to its nature as an electron donor. Protein pull-down assays revealed that CybE probably participates in metabolism and transfer of lipids, construction of cytoskeleton and mitochondria-associated energy metabolism to maintain the SRD accumulation in hyphal tips, membrane fluidity and hyphal extension. Findings in this study give a hint that inhibition of CybE may be an effective strategy for resisting the infection of the human pathogen A. fumigatus. Importance Investigating the knowledge of the growth regulation in the human opportunistic pathogen A. fumigatus is conducive to design new antifungal approach. The electron donor cytochrome b5 (CybE) plays a crucial role in maintaining the normal growth of A. fumigatus, however, the potential mechanism remains elusive. Herein, we characterized the molecular features of CybE and found the C-terminus with two transmembrane domains are required for its ER localization and functions. In addition, we demonstrated that CprA, an electron donor-heme-independent P450 reductase, provides a reciprocal function for the missing cytochrome b5 protein-CybE in A. fumigatus. CybE maintains the normal growth probably via supporting two crucial physiological processes, the SRD accumulation in hyphal tips and membrane fluidity. Therefore, our finding reveals the mechanisms underlying the regulatory effect of CybE on A. fumigatus growth and indicates that inhibition of CybE might be an effective approach for alleviating A. fumigatus infection.

Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain

2021 ◽  
Author(s):  
Marion Aruanno ◽  
Samantha Gozel ◽  
Isabelle Mouyna ◽  
Josie E Parker ◽  
Daniel Bachmann ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Fumigatus ◽  
Molecular Mechanisms ◽  
Drug Transporters ◽  
Major Facilitator Superfamily ◽  
Ergosterol Biosynthesis ◽  
Azole Resistance ◽  
Drug Transporter

Abstract Aspergillus fumigatus is the main cause of invasive aspergillosis, for which azole drugs are the first-line therapy. Emergence of pan-azole resistance among A. fumigatus is concerning and has been mainly attributed to mutations in the target gene (cyp51A). However, azole resistance may also result from other mutations (hmg1, hapE) or other adaptive mechanisms. We performed microevolution experiment exposing an A. fumigatus azole-susceptible strain (Ku80) to sub-minimal inhibitory concentration of voriconazole to analyze emergence of azole resistance. We obtained a strain with pan-azole resistance (Ku80R), which was partially reversible after drug relief, and without mutations in cyp51A, hmg1, and hapE. Transcriptomic analyses revealed overexpression of the transcription factor asg1, several ATP-binding cassette (ABC) and major facilitator superfamily transporters and genes of the ergosterol biosynthesis pathway in Ku80R. Sterol analysis showed a significant decrease of the ergosterol mass under voriconazole exposure in Ku80, but not in Ku80R. However, the proportion of the sterol compounds was similar between both strains. To further assess the role of transporters, we used the ABC transporter inhibitor milbemycine oxime (MLB). MLB inhibited transporter activity in both Ku80 and Ku80R and demonstrated some potentiating effect on azole activity. Criteria for synergism were reached for MLB and posaconazole against Ku80. Finally, deletion of asg1 revealed some role of this transcription factor in controlling drug transporter expression, but had no impact on azole susceptibility. This work provides further insight in mechanisms of azole stress adaptation and suggests that drug transporters inhibition may represent a novel therapeutic target. Lay Summary A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

scholarly journals SREBP-Dependent Triazole Susceptibility in Aspergillus fumigatus Is Mediated through Direct Transcriptional Regulation oferg11A(cyp51A)

2011 ◽  
Vol 56 (1) ◽  
pp. 248-257 ◽  
Author(s):  
Sara J. Blosser ◽  
Robert A. Cramer
Keyword(s):  
Aspergillus Fumigatus ◽  
Regulatory Element ◽  
Critical Role ◽  
Clinical Importance ◽  
Growth Defect ◽  
Ergosterol Biosynthesis ◽  
Wild Type ◽  
Transcript Levels ◽  
Content Type ◽  
Conditional Expression

ABSTRACTAs triazole antifungal drug resistance during invasiveAspergillus fumigatusinfection has become more prevalent, the need to understand mechanisms of resistance inA. fumigatushas increased. The presence of twoerg11(cyp51) genes inAspergillusspp. is hypothesized to account for the inherent resistance of this mold to the triazole fluconazole (FLC). Recently, anA. fumigatusnull mutant of a transcriptional regulator in the sterol regulatory element binding protein (SREBP) family, the ΔsrbAstrain, was found to have increased susceptibility to FLC and voriconazole (VCZ). In this study, we examined the mechanism engendering the observed increase inA. fumigatustriazole susceptibility in the absence of SrbA. We observed a significant reduction in theerg11Atranscript in the ΔsrbAstrain in response to FLC and VCZ. Transcript levels oferg11Bwere also reduced but not to the extent oferg11A. Interestingly,erg11Atranscript levels increased upon extended VCZ, but not FLC, exposure. Construction of anerg11Aconditional expression strain in the ΔsrbAstrain was able to restoreerg11Atranscript levels and, consequently, wild-type MICs to the triazole FLC. The VCZ MIC was also partially restored upon increasederg11Atranscript levels; however, total ergosterol levels remained significantly reduced compared to those of the wild type. Induction of theerg11Aconditional strain did not restore the hypoxia growth defect of the ΔsrbAstrain. Taken together, our results demonstrate a critical role for SrbA-mediated regulation of ergosterol biosynthesis and triazole drug interactions inA. fumigatusthat may have clinical importance.

Ergosterol biosynthesis pathway in Aspergillus fumigatus

Steroids ◽  
2008 ◽  
Vol 73 (3) ◽  
pp. 339-347 ◽  
Author(s):  
Laura Alcazar-Fuoli ◽  
Emilia Mellado ◽  
Guillermo Garcia-Effron ◽  
Jordi F. Lopez ◽  
Joan O. Grimalt ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Ergosterol Biosynthesis ◽  
Biosynthesis Pathway

scholarly journals Erg4A and Erg4B Are Required for Conidiation and Azole Resistance via Regulation of Ergosterol Biosynthesis in Aspergillus fumigatus

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Nanbiao Long ◽  
Xiaoling Xu ◽  
Qiuqiong Zeng ◽  
Hong Sang ◽  
Ling Lu
Keyword(s):  
Endoplasmic Reticulum ◽  
Aspergillus Fumigatus ◽  
Plasma Membranes ◽  
Current Knowledge ◽  
Hyphal Growth ◽  
Antifungal Drugs ◽  
Ergosterol Biosynthesis ◽  
Wild Type ◽  
Content Type ◽  
Ergosterol Synthesis

ABSTRACT Ergosterol, a fungus-specific sterol enriched in cell plasma membranes, is an effective antifungal drug target. However, current knowledge of the ergosterol biosynthesis process in the saprophytic human fungal pathogen Aspergillus fumigatus remains limited. In this study, we found that two endoplasmic reticulum-localized sterol C-24 reductases encoded by both erg4A and erg4B homologs are required to catalyze the reaction during the final step of ergosterol biosynthesis. Loss of one homolog of Erg4 induces the overexpression of the other one, accompanied by almost normal ergosterol synthesis and wild-type colony growth. However, double deletions of erg4A and erg4B completely block the last step of ergosterol synthesis, resulting in the accumulation of ergosta-5,7,22,24(28)-tetraenol, a precursor compound of ergosterol. Further studies indicate that erg4A and erg4B are required for conidiation but not for hyphal growth. Importantly, the Δerg4A Δerg4B mutant still demonstrates wild-type virulence in a compromised mouse model but displays remarkable increased susceptibility to antifungal azoles. Our data suggest that inhibitors of Erg4A and Erg4B may serve as effective candidates for adjunct antifungal agents with azoles. IMPORTANCE Knowledge of the ergosterol biosynthesis pathway in the human opportunistic pathogen A. fumigatus is useful for designing and finding new antifungal drugs. In this study, we demonstrated that the endoplasmic reticulum-localized sterol C-24 reductases Erg4A and Erg4B are required for conidiation via regulation of ergosterol biosynthesis. Moreover, inactivation of both Erg4A and Erg4B results in hypersensitivity to the clinical guideline-recommended antifungal drugs itraconazole and voriconazole. Therefore, our finding indicates that inhibition of Erg4A and Erg4B might be an effective approach for alleviating A. fumigatus infection.

scholarly journals Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2492-2506 ◽  
Author(s):  
Sara J. Blosser ◽  
Brittney Merriman ◽  
Nora Grahl ◽  
Dawoon Chung ◽  
Robert A. Cramer
Keyword(s):  
Aspergillus Fumigatus ◽  
Growth Defect ◽  
Ergosterol Biosynthesis ◽  
Mammalian Host ◽  
Primary Role ◽  
Single Deletion ◽  
Methyl Sterol ◽  
Genes Encoding ◽  
Methyl Sterols

The human pathogen Aspergillus fumigatus adapts to stress encountered in the mammalian host as part of its ability to cause disease. The transcription factor SrbA plays a significant role in this process by regulating genes involved in hypoxia and low-iron adaptation, antifungal drug responses and virulence. SrbA is a direct transcriptional regulator of genes encoding key enzymes in the ergosterol biosynthesis pathway, including erg25A and erg25B, and ΔsrbA accumulates C4-methyl sterols, suggesting a loss of Erg25 activity [C4-sterol methyl oxidase (SMO)]. Characterization of the two genes encoding SMOs in Aspergillus fumigatus revealed that both serve as functional C4-demethylases, with Erg25A serving in a primary role, as Δerg25A accumulates more C4-methyl sterol intermediates than Δerg25B. Single deletion of these SMOs revealed alterations in canonical ergosterol biosynthesis, indicating that ergosterol may be produced in an alternative fashion in the absence of SMO activity. A Δerg25A strain displayed moderate susceptibility to hypoxia and the endoplasmic reticulum stress-inducing agent DTT, but was not required for virulence in murine or insect models of invasive aspergillosis. Inducing expression of erg25A partially restored the hypoxia growth defect of ΔsrbA. These findings implicated Aspergillus fumigatus SMOs in the maintenance of canonical ergosterol biosynthesis and indicated an overall involvement in the fungal stress response.

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 Aspergillus fumigatus C-5 Sterol Desaturases Erg3A and Erg3B: Role in Sterol Biosynthesis and Antifungal Drug Susceptibility

2006 ◽  
Vol 50 (2) ◽  
pp. 453-460 ◽  
Author(s):  
Laura Alcazar-Fuoli ◽  
Emilia Mellado ◽  
Guillermo Garcia-Effron ◽  
Maria J. Buitrago ◽  
Jordi F. Lopez ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Amphotericin B ◽  
Gene Knockout ◽  
Drug Susceptibility ◽  
Nucleotide Sequences ◽  
Ergosterol Biosynthesis ◽  
Null Mutant ◽  
Targeted Disruption ◽  
Genes Encoding ◽  
Mutant Phenotypes

ABSTRACT Two erg3 genes encoding C-5 sterol desaturase enzymes (Erg3A and Erg3B) in Aspergillus fumigatus were characterized with respect to their nucleotide sequences and null mutant phenotypes. Targeted disruption of the erg3A and erg3B genes and a double gene knockout, erg3A − erg3B −, showed that they are not essential for A. fumigatus viability. Mutant phenotypes clearly showed that Erg3B is a C-5 sterol desaturase, but no apparent role for Erg3A in A. fumigatus ergosterol biosynthesis was found. Susceptibility to amphotericin B, itraconazole, fluconazole, voriconazole, and ketoconazole was not altered in isolates in which erg3A and erg3B were knocked out alone and in combination.

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