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

Abstract

Ergosterol, a major lipid present in the fungal cell membrane, is considered as an effective antifungal drug target. A rational strategy for increasing drug reservoir relies on functionally validation of essential enzymes involved in fungal key biological pathway. Current knowledge regarding the essential genes in the ergosterol biosynthesis pathway is still limited in the opportunistic human pathogen Aspergillus fumigatus. In this study, we characterized two endoplasmic reticulum-localized sterol C-14 reductases encoded by both erg24A and erg24B homologs that are essential for the viability of A. fumigatus despite the fact that neither paralog is essential individually. Loss of one homolog of Erg24 impairs hyphal growth, conidiation, and virulence but has no effect on ergosterol biosynthesis. To investigate the functional significance of erg24, a conditional double mutant (Δerg24B niiA::erg24A) was constructed in the Δerg24B background. Strikingly, the conditional erg24 double mutant exhibited severe growth defects and accumulation of sterol intermediate. Moreover, the addition of metal ions and the overexpression of the corresponding ion transporters could rescue the growth defects of the erg24 double mutant in A. fumigatus, implying that the defective phenotype of the erg24 double mutant is tightly associated with dysregulation of ion homeostasis. Taken together, our results demonstrate the critical role of Erg24 in ergosterol biosynthesis and ion homeostasis in A. fumigatus, which may have important implications for antifungal discovery.

Key points

We characterized two endoplasmic reticulum-localized sterol C-14 reductases Erg24A and Erg24B in A. fumigatus.

Erg24A and Erg24B in combination, but not individually, are required for the viability of A. fumigatus.

Inactivation of Erg24 leads to the disruption of ion homeostasis and affects ergosterol biosynthesis.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Data availability

Not applicable.

References

  1. Abad A, Fernandez-Molina JV, Bikandi J, Ramirez A, Margareto J, Sendino J, Hernando FL, Ponton J, Garaizar J, Rementeria A (2010) What makes Aspergillus fumigatus a successful pathogen? Genes and molecules involved in invasive aspergillosis. Rev Iberoam Micol 27(4):155–182. https://doi.org/10.1016/j.riam.2010.10.003

    Article  PubMed  Google Scholar 

  2. Alcazar-Fuoli L, Mellado E, Garcia-Effron G, Buitrago MJ, Lopez JF, Grimalt JO, Cuenca-Estrella JM, Rodriguez-Tudela JL (2006) Aspergillus fumigatus C-5 sterol desaturases Erg3A and Erg3B: role in sterol biosynthesis and antifungal drug susceptibility. Antimicrob Agents Chemother 50(2):453–460. https://doi.org/10.1128/AAC.50.2.453-460.2006

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Alcazar-Fuoli L, Mellado E, Garcia-Effron G, Lopez JF, Grimalt JO, Cuenca-Estrella JM, Rodriguez-Tudela JL (2008) Ergosterol biosynthesis pathway in Aspergillus fumigatus. Steroids 73(3):339–347. https://doi.org/10.1016/j.steroids.2007.11.005

    CAS  Article  PubMed  Google Scholar 

  4. Amaar YG, Moore MM (1998) Mapping of the nitrate-assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus. Curr Genet 33(3):206–215. https://doi.org/10.1007/s002940050328

    CAS  Article  PubMed  Google Scholar 

  5. Anderson TM, Clay MC, Cioffi AG, Diaz KA, Hisao GS, Tuttle MD, Nieuwkoop AJ, Comellas G, Maryum N, Wang S, Uno BE, Wildeman EL, Gonen T, Rienstra CM, Burke MD (2014) Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat Chem Biol 10(5):400–406. https://doi.org/10.1038/nchembio.1496

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Athanasopoulos A, Andre B, Sophianopoulou V, Gournas C (2019) Fungal plasma membrane domains. FEMS Microbiol Rev 43(6):642–673. https://doi.org/10.1093/femsre/fuz022

    CAS  Article  PubMed  Google Scholar 

  7. Blatzer M, Latge JP (2017) Metal-homeostasis in the pathobiology of the opportunistic human fungal pathogen Aspergillus fumigatus. Curr Opin Microbiol 40:152–159. https://doi.org/10.1016/j.mib.2017.11.015

    CAS  Article  PubMed  Google Scholar 

  8. Blosser SJ, Merriman B, Grahl N, Chung D, Cramer RA (2014) Two C4-sterol methyl oxidases (Erg25) catalyse ergosterol intermediate demethylation and impact environmental stress adaptation in Aspergillus fumigatus. Microbiology (Reading) 160(Pt 11):2492–2506. https://doi.org/10.1099/mic.0.080440-0

    CAS  Article  Google Scholar 

  9. Brakhage AA, Langfelder K (2002) Menacing mold: the molecular biology of Aspergillus fumigatus. Annu Rev Microbiol 56:433–455. https://doi.org/10.1146/annurev.micro.56.012302.160625

    CAS  Article  PubMed  Google Scholar 

  10. Crowley JH, Smith SJ, Leak FW, Parks LW (1996) Aerobic isolation of an ERG24 null mutant of Saccharomyces cerevisiae. J Bacteriol 178(10):2991–2993. https://doi.org/10.1128/jb.178.10.2991-2993.1996

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Crowley JH, Tove S, Parks LW (1998) A calcium-dependent ergosterol mutant of Saccharomyces cerevisiae. Curr Genet 34(2):93–99. https://doi.org/10.1007/s002940050371

    CAS  Article  PubMed  Google Scholar 

  12. Cyert MS, Philpott CC (2013) Regulation of cation balance in Saccharomyces cerevisiae. Genetics 193(3):677–713. https://doi.org/10.1534/genetics.112.147207

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Dhingra S, Cramer RA (2017) Regulation of Sterol Biosynthesis in the Human Fungal Pathogen Aspergillus fumigatus: Opportunities for Therapeutic Development. Front Microbiol 8:92. https://doi.org/10.3389/fmicb.2017.00092

    Article  PubMed  PubMed Central  Google Scholar 

  14. Fallon JP, Troy N, Kavanagh K (2011) Pre-exposure of Galleria mellonella larvae to different doses of Aspergillus fumigatus conidia causes differential activation of cellular and humoral immune responses. Virulence 2(5):413–421. https://doi.org/10.4161/viru.2.5.17811

    Article  PubMed  Google Scholar 

  15. Francois JM (2016) Cell Surface Interference with Plasma Membrane and Transport Processes in Yeasts. Adv Exp Med Biol 892:11–31. https://doi.org/10.1007/978-3-319-25304-6_2

    CAS  Article  PubMed  Google Scholar 

  16. Gerwien F, Skrahina V, Kasper L, Hube B, Brunke S (2018) Metals in fungal virulence. FEMS Microbiol Rev 42(1). https://doi.org/10.1093/femsre/fux050

  17. Hu W, Sillaots S, Lemieux S, Davison J, Kauffman S, Breton A, Linteau A, Xin C, Bowman J, Becker J, Jiang B, Roemer T (2007) Essential gene identification and drug target prioritization in Aspergillus fumigatus. PLoS Pathog 3(3):e24. https://doi.org/10.1371/journal.ppat.0030024

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Jia N, Arthington-Skaggs B, Lee W, Pierson CA, Lees ND, Eckstein J, Barbuch R, Bard M (2002) Candida albicans sterol C-14 reductase, encoded by the ERG24 gene, as a potential antifungal target site. Antimicrob Agents Chemother 46(4):947–957. https://doi.org/10.1128/aac.46.4.947-957.2002

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Jiang H, Shen Y, Liu W, Lu L (2014) Deletion of the putative stretch-activated ion channel Mid1 is hypervirulent in Aspergillus fumigatus. Fungal Genet Biol 62:62–70. https://doi.org/10.1016/j.fgb.2013.11.003

    CAS  Article  PubMed  Google Scholar 

  20. Jorda T, Puig S (2020) Regulation of Ergosterol Biosynthesis in Saccharomyces cerevisiae. Genes (Basel) 11(7). https://doi.org/10.3390/genes11070795

  21. Latge JP (1999) Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12(2):310–350

    CAS  Article  Google Scholar 

  22. Latge JP (2001) The pathobiology of Aspergillus fumigatus. Trends Microbiol 9(8):382–389. https://doi.org/10.1016/s0966-842x(01)02104-7

    CAS  Article  PubMed  Google Scholar 

  23. Li Y, Zhang Y, Zhang C, Wang H, Wei X, Chen P, Lu L (2020) Mitochondrial dysfunctions trigger the calcium signaling-dependent fungal multidrug resistance. Proc Natl Acad Sci U S A 117(3):1711–1721. https://doi.org/10.1073/pnas.1911560116

    CAS  Article  PubMed  Google Scholar 

  24. Liu X, Fu J, Yun Y, Yin Y, Ma Z (2011) A sterol C-14 reductase encoded by FgERG24B is responsible for the intrinsic resistance of Fusarium graminearum to amine fungicides. Microbiology (Reading) 157(Pt 6):1665–1675. https://doi.org/10.1099/mic.0.045690-0

    CAS  Article  Google Scholar 

  25. Liu JF, Xia JJ, Nie KL, Wang F, Deng L (2019) Outline of the biosynthesis and regulation of ergosterol in yeast. World J Microbiol Biotechnol 35(7):98. https://doi.org/10.1007/s11274-019-2673-2

    Article  PubMed  Google Scholar 

  26. Long N, Xu X, Zeng Q, Sang H, Lu L (2017) Erg4A and Erg4B Are Required for Conidiation and Azole Resistance via Regulation of Ergosterol Biosynthesis in Aspergillus fumigatus. Appl Environ Microbiol 83(4). https://doi.org/10.1128/AEM.02924-16

  27. Lorenz RT, Parks LW (1992) Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae. DNA Cell Biol 11(9):685–692. https://doi.org/10.1089/dna.1992.11.685

    CAS  Article  PubMed  Google Scholar 

  28. Luna-Tapia A, Peters BM, Eberle KE, Kerns ME, Foster TP, Marrero L, Noverr MC, Fidel PL Jr, Palmer GE (2015) ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis. Eukaryot Cell 14(10):1006–1016. https://doi.org/10.1128/EC.00116-15

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Lupetti A, Danesi R, Campa M, Del Tacca M, Kelly S (2002) Molecular basis of resistance to azole antifungals. Trends Mol Med 8(2):76–81. https://doi.org/10.1016/s1471-4914(02)02280-3

    CAS  Article  PubMed  Google Scholar 

  30. Maxfield FR, Tabas I (2005) Role of cholesterol and lipid organization in disease. Nature 438(7068):612–621. https://doi.org/10.1038/nature04399

    CAS  Article  PubMed  Google Scholar 

  31. May GS (1989) The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable. J Cell Biol 109(5):2267–2274. https://doi.org/10.1083/jcb.109.5.2267

    CAS  Article  PubMed  Google Scholar 

  32. Muro-Pastor MI, Gonzalez R, Strauss J, Narendja F, Scazzocchio C (1999) The GATA factor AreA is essential for chromatin remodelling in a eukaryotic bidirectional promoter. EMBO J 18(6):1584–1597. https://doi.org/10.1093/emboj/18.6.1584

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Nelson G, Kozlova-Zwinderman O, Collis AJ, Knight MR, Fincham JR, Stanger CP, Renwick A, Hessing JG, Punt PJ, van den Hondel CA, Read ND (2004) Calcium measurement in living filamentous fungi expressing codon-optimized aequorin. Mol Microbiol 52(5):1437–1450. https://doi.org/10.1111/j.1365-2958.2004.04066.x

    CAS  Article  PubMed  Google Scholar 

  34. Odds FC, Brown AJ, Gow NA (2003) Antifungal agents: mechanisms of action. Trends Microbiol 11(6):272–279. https://doi.org/10.1016/s0966-842x(03)00117-3

    CAS  Article  PubMed  Google Scholar 

  35. O'Gorman CM, Fuller H, Dyer PS (2009) Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus. Nature 457(7228):471–474. https://doi.org/10.1038/nature07528

    CAS  Article  PubMed  Google Scholar 

  36. Osmani SA, Pu RT, Morris NR (1988) Mitotic induction and maintenance by overexpression of a G2-specific gene that encodes a potential protein kinase. Cell 53(2):237–244

    CAS  Article  Google Scholar 

  37. Palacios DS, Dailey I, Siebert DM, Wilcock BC, Burke MD (2011) Synthesis-enabled functional group deletions reveal key underpinnings of amphotericin B ion channel and antifungal activities. Proc Natl Acad Sci U S A 108(17):6733–6738. https://doi.org/10.1073/pnas.1015023108

    Article  PubMed  PubMed Central  Google Scholar 

  38. Perlin DS, Shor E, Zhao Y (2015) Update on Antifungal Drug Resistance. Curr Clin Microbiol Rep 2(2):84–95. https://doi.org/10.1007/s40588-015-0015-1

    Article  PubMed  PubMed Central  Google Scholar 

  39. Pinchai N, Juvvadi PR, Fortwendel JR, Perfect BZ, Rogg LE, Asfaw YG, Steinbach WJ (2010) The Aspergillus fumigatus P-type Golgi apparatus Ca2+/Mn2+ ATPase PmrA is involved in cation homeostasis and cell wall integrity but is not essential for pathogenesis. Eukaryot Cell 9(3):472–476. https://doi.org/10.1128/EC.00378-09

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. Robbins N, Caplan T, Cowen LE (2017) Molecular evolution of antifungal drug resistance. Annu Rev Microbiol 71:753–775. https://doi.org/10.1146/annurev-micro-030117-020345

    CAS  Article  PubMed  Google Scholar 

  41. Roemer T, Krysan DJ (2014) Antifungal drug development: challenges, unmet clinical needs, and new approaches. Cold Spring Harb Perspect Med 4(5). https://doi.org/10.1101/cshperspect.a019703

  42. Sant DG, Tupe SG, Ramana CV, Deshpande MV (2016) Fungal cell membrane-promising drug target for antifungal therapy. J Appl Microbiol 121(6):1498–1510. https://doi.org/10.1111/jam.13301

    CAS  Article  PubMed  Google Scholar 

  43. Schiraldi GF, Colombo MD, Harari S, Lo Cicero S, Ziglio G, Ferrarese M, Rossato D, Soresi E (1996) Terbinafine in the treatment of non-immunocompromised compassionate cases of bronchopulmonary aspergillosis. Mycoses 39(1-2):5–12. https://doi.org/10.1111/j.1439-0507.1996.tb00077.x

    CAS  Article  PubMed  Google Scholar 

  44. Shah Alam Bhuiyan M, Eckstein J, Barbuch R, Bard M (2007) Synthetically lethal interactions involving loss of the yeast ERG24: the sterol C-14 reductase gene. Lipids 42(1):69–76. https://doi.org/10.1007/s11745-006-1001-4

    CAS  Article  PubMed  Google Scholar 

  45. Song J, Liu X, Li R (2020) Sphingolipids: Regulators of azole drug resistance and fungal pathogenicity. Mol Microbiol 114:891–905. https://doi.org/10.1111/mmi.14586

    CAS  Article  PubMed  Google Scholar 

  46. Tada R, Latge JP, Aimanianda V (2013) Undressing the fungal cell wall/cell membrane--the antifungal drug targets. Curr Pharm Des 19(20):3738–3747. https://doi.org/10.2174/1381612811319200012

    CAS  Article  PubMed  Google Scholar 

  47. Tekaia F, Latge JP (2005) Aspergillus fumigatus: saprophyte or pathogen? Curr Opin Microbiol 8(4):385–392. https://doi.org/10.1016/j.mib.2005.06.017

    CAS  Article  PubMed  Google Scholar 

  48. Upadhyay S, Torres G, Lin X (2013) Laccases involved in 1,8-dihydroxynaphthalene melanin biosynthesis in Aspergillus fumigatus are regulated by developmental factors and copper homeostasis. Eukaryot Cell 12(12):1641–1652. https://doi.org/10.1128/EC.00217-13

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. van de Veerdonk FL, Gresnigt MS, Romani L, Netea MG, Latge JP (2017) Aspergillus fumigatus morphology and dynamic host interactions. Nat Rev Microbiol 15(11):661–674. https://doi.org/10.1038/nrmicro.2017.90

    CAS  Article  PubMed  Google Scholar 

  50. Zhang YQ, Gamarra S, Garcia-Effron G, Park S, Perlin DS, Rao R (2010) Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs. PLoS Pathog 6(6):e1000939. https://doi.org/10.1371/journal.ppat.1000939

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Zhang C, Meng X, Wei X, Lu L (2016) Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet Biol 86:47–57. https://doi.org/10.1016/j.fgb.2015.12.007

    CAS  Article  PubMed  Google Scholar 

  52. Zhang Y, Wei W, Fan J, Jin C, Lu L, Fang W (2020) Aspergillus fumigatus Mitochondrial Acetyl Coenzyme A Acetyltransferase as an Antifungal Target. Appl Environ Microbiol 86(7). https://doi.org/10.1128/AEM.02986-19

Download references

Acknowledgments

We thank Dr. Chi Zhang for kindly helping the construction of strains.

Funding

This work was financially supported by grants from the National Key Research and Development Program of China (2019YFA0904900), the National Natural Science Foundation of China (31861133014 and 31770086), the Program for Jiangsu Excellent Scientific and Technological Innovation team (17CXTD00014), the Priority Academic Program Development of Jiangsu Higher Education Institutions to L.L, and the National Natural Science Foundation of China (31900404), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (19KJB180017) to Y.Z.

Author information

Affiliations

Authors

Contributions

YL and LL conceived and designed research; YL and MD conducted experiments. YL, YZ, and LL analyzed data. YL, YZ, and LL wrote the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Yuanwei Zhang or Ling Lu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

ESM 1

(PDF 9925 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, Y., Dai, M., Zhang, Y. et al. The sterol C-14 reductase Erg24 is responsible for ergosterol biosynthesis and ion homeostasis in Aspergillus fumigatus. Appl Microbiol Biotechnol 105, 1253–1268 (2021). https://doi.org/10.1007/s00253-021-11104-5

Download citation

Keywords

  • Ergosterol biosynthesis
  • Ion homeostasis
  • Aspergillus fumigatus
  • Sterol C-14 reductase