Current Genetics

, Volume 64, Issue 5, pp 1043–1056 | Cite as

Protein O-mannosyltransferases are required for sterigmatocystin production and developmental processes in Aspergillus nidulans

  • Thi Huynh Tram Le
  • Ayana Oki
  • Masatoshi Goto
  • Kiminori ShimizuEmail author
Original Article


Aspergillus nidulans produces sterigmatocystin (ST), a precursor of a carcinogenic secondary metabolite aflatoxin (AF), during its developmental process. ST biosynthesis has been shown to be affected by various regulatory factors. In this study, we investigated the involvement of O-mannosyltransferases (PmtA, PmtB, PmtC), in ST production and morphological development. Deletion of pmtApmtA), pmtBpmtB) or pmtCpmtC) caused no spore production and a significant decline of vegetative growth. A tremendous decline of ST level was observed in all Δpmt mutants at the third day after inoculation. By extending the growth period, ST production of ΔpmtA and ΔpmtB increased to the wild-type level 7 days after inoculation. On the other hand, ST was not detected from 7- or 14-day cultures in ΔpmtC. Expression levels of aflR gene, an essential regulator of the ST biosynthesis pathway, were also down-regulated in the Δpmt strains. By introducing the aflR overexpression cassette, ST production in the ΔpmtA and ΔpmtB significantly increased to levels comparable to the wild type. However, the presence of the aflR overexpression cassette could not improve ST production in the ΔpmtC mutant. These data suggest that the PMT family is a new endogenous factor that is required for ST biosynthesis in A. nidulans. These findings provide better understanding of the regulatory mechanisms of AF/ST biosynthesis, which can ultimately contribute to our ability to control aflatoxin contamination.


AflR Pmt Regulation Secondary metabolite 



We are grateful to Nancy Keller of the University of Wisconsin–Madison for strains and Heng Chooi of the University of Western Australia for the plasmid pYellow. We thank Darby Brown for reviewing the manuscript before submission. This research was financially supported by the Intramural Startup Funding from the Tokyo University of Science.

Supplementary material

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Supplementary material 1 (DOCX 44 KB)
294_2018_816_MOESM2_ESM.docx (44 kb)
Supplementary material 2 (DOCX 44 KB)
294_2018_816_MOESM3_ESM.docx (72 kb)
Supplementary material 3 (DOCX 72 KB)


  1. Abu-Qarn M, Eichler J, Sharon N (2008) Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea. Curr Opin Struct Biol 18:544–550CrossRefPubMedCentralGoogle Scholar
  2. Ahmed AM, Tabana Y, Musa K, Sandai D (2017) Effects of different mycotoxins on humans, cell genome and their involvement in cancer (review). Oncol Rep 37:1321–1336CrossRefGoogle Scholar
  3. Alam MA, Kamlangdee N, Kelly JM (2017) The CreB deubiquitinating enzyme does not directly target the CreA repressor protein in Aspergillus nidulans. Curr Genet 63:647–667CrossRefPubMedCentralGoogle Scholar
  4. Bayram Ö, Bayram ÖS, Ahmed YL, Maruyama J, Valerius O, Rizzoli SO, Ficner R, Irniger S, Braus GH (2012) The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism. PLoS Genet 8:1–19CrossRefGoogle Scholar
  5. Bayram ÖS, Latgé JP, Bayram Ö (2017) MybA, a new player driving survival of the conidium of the human pathogen Aspergillus fumigatus. Curr Genet  64(1):141–146CrossRefPubMedCentralGoogle Scholar
  6. Bennett JW, Klich M (2003) Mycotoxins. Clin Microbiol Rev 16:497–516CrossRefPubMedCentralGoogle Scholar
  7. Brodhagen M, Keller NP (2006) Signalling pathways connecting mycotoxin production and sporulation. Mol Plant Pathol 7:285–301CrossRefPubMedCentralGoogle Scholar
  8. Brown DW, Yu JH, Kelkar HS, Fernandes M, Nesbitt TC, Keller NP, Adams TH, Leonard TJ (1996) Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans. Proc Natl Acad Sci USA 93:1418–1422CrossRefPubMedCentralGoogle Scholar
  9. Calvo A, Dhingra S (2011) Conserved regulatory mechanisms controlling aflatoxin and sterigmatocystin biosynthesis. In: Guevara-Gonzalez RG (ed) Aflatoxins—biochemistry and molecular biology. InTech, Rijeka, Croatia, pp 67–88Google Scholar
  10. Calvo AM, Wilson RA, Bok JW, Keller NP (2002) Relationship between secondary metabolism and fungal development. Microbiol Mol Biol Rev 66:447–459CrossRefPubMedCentralGoogle Scholar
  11. Calvo AM, Bok J, Brooks W, Keller NP (2004) veA is required for toxin and sclerotial production in Aspergillus parasiticus. Appl Environ Microbiol 70:4733–4739CrossRefPubMedCentralGoogle Scholar
  12. Chu XL, Feng MG, Ying SH (2016) Qualitative ubiquitome unveils the potential significances of protein lysine ubiquitination in hyphal growth of Aspergillus nidulans. Curr Genet 62:191–201CrossRefPubMedCentralGoogle Scholar
  13. Cygler M, Schrag JD, Sussman JL, Harel M, Silman I, Gentry MK, Doctor BP (1993) Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins. Protein Sci 2:366–382CrossRefPubMedCentralGoogle Scholar
  14. Dean N (1995) Yeast glycosylation mutants are sensitive to aminoglycosides. Proc Natl Acad Sci USA 92:1287–1291CrossRefPubMedCentralGoogle Scholar
  15. Duran RM, Cary JW, Calvo AM (2006) Production of cyclopiazonic acid, aflatrem, and aflatoxin by Aspergillus flavus is regulated by veA, a gene necessary for sclerotial formation. Appl Microbiol Biotechnol 73:1158–1168CrossRefPubMedCentralGoogle Scholar
  16. Fang W, Ding W, Wang B, Zhou H, Ouyang H, Ming J, Jin C (2010) Reduced expression of the O-mannosyltransferase 2 (AfPmt2) leads to deficient cell wall and abnormal polarity in Aspergillus fumigatus. Glycobiology 20:542–552CrossRefPubMedCentralGoogle Scholar
  17. Feng GH, Leonard TJ (1998) Culture conditions control expression of the genes for aflatoxin and sterigmatocystin biosynthesis in Aspergillus parasiticus and A. nidulans. Appl Environ Microbiol 64:2275–2277PubMedPubMedCentralGoogle Scholar
  18. Flaherty JE, Payne GA (1997) Overexpression of aflR leads to upregulation of pathway gene expression and increased aflatoxin production in Aspergillus flavus. Appl Environ Microbiol 63:3995–4000PubMedPubMedCentralGoogle Scholar
  19. Futagami T, Nakao S, Kido Y, Oka T, Kajiwara Y, Takashita H, Omori T, Furukawa K, Goto M (2011) Putative stress sensors WscA and WscB are involved in hypo-osmotic and acidic pH stress tolerance in Aspergillus nidulans. Eukaryot Cell 10:1504–1515CrossRefPubMedCentralGoogle Scholar
  20. Gentzsch M, Tanner W (1996) The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital. EMBO J 15:5752–5759CrossRefPubMedCentralGoogle Scholar
  21. Gentzsch M, Tanner W, Seidl T (1997) Protein O-glycosylation in yeast: protein-specific mannosyltransferases. Glycobiology 7:481–486CrossRefPubMedCentralGoogle Scholar
  22. Girrbach V, Zeller T, Priesmeier M, Strahl-Bolsinger S (2000) Structure-function analysis of the dolichyl phosphate-mannose: protein O-mannosyltransferase ScPmt1p. J Biol Chem 275:19288–19296CrossRefPubMedCentralGoogle Scholar
  23. Goto M, Harada Y, Oka T, Matsumoto S, Takegawa K, Furukawa K (2009) Protein O-mannosyltransferases B and C support hyphal development and differentiation in Aspergillus nidulans. Eukaryot Cell 8:1465–1474CrossRefPubMedCentralGoogle Scholar
  24. Guzmán-de-Peña D, Ruiz-Herrera J (1997) Relationship between aflatoxin biosynthesis and sporulation in Aspergillus parasiticus. Fungal Genet Biol 21:198–205CrossRefPubMedCentralGoogle Scholar
  25. Hajjar JD, Bennett JW, Bhatnagar D, Bahu R (1989) Sterigmatocystin production by laboratory strains of Aspergillus nidulans. Mycol Res 93:548–551CrossRefGoogle Scholar
  26. Hussein H, Brasel J (2001) Toxicity, metabolism and impact of mycotoxins on human and animals. Toxicology 167:101–134CrossRefPubMedCentralGoogle Scholar
  27. Kato N, Brooks W, Calvo AM (2003) The expression of sterigmatocystin and penicillin genes in Aspergillus nidulans is controlled by veA, a gene required for sexual development. Am Soc Microbiol 2:1178–1186Google Scholar
  28. Keller NP, Adams TH (1995) Analysis of a mycotoxin gene cluster in Aspergillus nidulans. SAAS Bull Biochem Biotechnol 8:14–21PubMedPubMedCentralGoogle Scholar
  29. Kriangkripipat T, Momany M (2009) Aspergillus nidulans protein O-mannosyltransferases play roles in cell wall integrity and developmental patterning. Eukaryot Cell 8:1475–1485CrossRefPubMedCentralGoogle Scholar
  30. Kriangkripipat T, Momany M (2014) Aspergillus nidulans Pmts form heterodimers in all pairwise combinations. FEBS Open Bio 4:335–341CrossRefPubMedCentralGoogle Scholar
  31. L∆iu BH, Chu FS (1998) Regulation of aflR and its product, AflR, associated with aflatoxin biosynthesis. Appl Environ Microbiol 64:3718–3723PubMedPubMedCentralGoogle Scholar
  32. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real- time quantitative PCR and the 2−∆∆CT method. Methods 25:402–408CrossRefPubMedCentralGoogle Scholar
  33. Lommel M, Bagnat M, Strahl S (2004) Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants. Mol Cell Biol 24:46–57CrossRefPubMedCentralGoogle Scholar
  34. Moretfl E, Segoviat L (1993) The sigma54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains. J Bacteriol 175:6067–6074CrossRefGoogle Scholar
  35. Mouyna I, Kniemeyer O, Jank T, Loussert C, Mellado E, Aimanianda V, Beauvais A, Wartenberg D, Sarfati J, Bayry J, Prévost MC, Brakhage AA, Strahl S, Huerre M, Latgé JP (2010) Members of protein O-mannosyltransferase family in Aspergillus fumigatus differentially affect growth, morphogenesis and viability. Mol Microbiol 76:1205–1221CrossRefPubMedCentralGoogle Scholar
  36. Oiartzabal-Arano E, Perez-de-Nanclares-Arregi E, Espeso EA, Etxebeste O (2016) Apical control of conidiation in Aspergillus nidulans. Curr Genet 62:371–377CrossRefPubMedCentralGoogle Scholar
  37. Oka T, Hamaguchi T, Sameshima Y, Goto M, Furukawa K (2004) Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans. Microbiology 150:1973–1982CrossRefPubMedCentralGoogle Scholar
  38. Oka T, Sameshima Y, Koga T, Kim H, Goto M, Furukawa K (2005) Protein O-mannosyltransferase A of Aspergillus awamori is involved in O-mannosylation of glucoamylase I. Microbiology 151:3657–3667CrossRefPubMedCentralGoogle Scholar
  39. Olson GM, Fox DS, Wang P, Alspaugh JA, Buchanan KL (2007) Role of protein O-mannosyltransferase Pmt4 in the morphogenesis and virulence of Cryptococcus neoformans. Eukaryot Cell 6:222–234CrossRefPubMedCentralGoogle Scholar
  40. Prill SKH, Klinkert B, Timpel C, Gale CA, Schröppel K, Ernst JF (2005) PMT family of Candida albicans: Five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance. Mol Microbiol 55:546–560CrossRefPubMedCentralGoogle Scholar
  41. Rouabhia M, Schaller M, Corbucci C, Vecchiarelli A, Prill SK, Giasson L, Ernst JF (2005) Virulence of the fungal pathogen Candida albicans requires the five isoforms of protein mannosyltransferases. Infect Immun 73:4571–4580CrossRefPubMedCentralGoogle Scholar
  42. Shimizu K, Keller NP (2001) Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics 157:591–600PubMedPubMedCentralGoogle Scholar
  43. Shimizu K, Imanishi Y, Toh-e A, Uno J, Chibana H, Hull CM, Kawamoto S (2014) Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans. Fungal Genet Biol 69:13–22CrossRefPubMedCentralGoogle Scholar
  44. Strahl_Bolsinger S, Immervoll T, Deutzmanni R, Tanner W (1993) PMT1, the gene for a key enzyme of protein O-glycosylation in Saccharomyces cerevisiae. Biochemistry 90:8164–8168Google Scholar
  45. Strahl-Bolsinger S, Tanner W (1991) Protein O-glycosylation in Saccharomyces cerevisiae. Purification and characterization of the dolichyl-phosphate-d-mannose-protein O-d-mannosyltransferase. Eur J Biochem 190:185–190CrossRefGoogle Scholar
  46. Strahl-Bolsinger S, Gentzsch M, Tanner W (1999) Protein O-mannosylation. Biochim Biophys Acta Gen Subj 1426:297–307CrossRefGoogle Scholar
  47. Timpel C, Zink S, Strahl-Bolsinger S, Schröppel K, Ernst J (2000) Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannosyltransferase in the fungal pathogen Candida albicans. J Bacteriol 182:3063–3071CrossRefPubMedCentralGoogle Scholar
  48. Verna J, Lodder A, Lee K, Vagts A, Ballester R (1997) A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 94:13804–13809CrossRefPubMedCentralGoogle Scholar
  49. Wilkinson HH, Ramaswamy A, Sim SC, Keller NP (2004) Increased conidiation associated with progression along the sterigmatocystin biosynthetic pathway. Mycologia 96:1190CrossRefPubMedCentralGoogle Scholar
  50. Willer T, Amselgruber W, Deutzmann R, Strahl S (2002) Characterization of POMT2, a novel member of the PMT protein O-mannosyltransferase family specifically localized to the acrosome of mammalian spermatids. Glycobiology 12:771–783CrossRefPubMedCentralGoogle Scholar
  51. Willer T, Brandl M, Sipiczki M, Strahl S (2005) Protein O-mannosylation is crucial for cell wall integrity, septation and viability in fission yeast. Mol Microbiol 57:156–170CrossRefPubMedCentralGoogle Scholar
  52. Willger SD, Ernst JF, Alspaugh JA, Lengeler KB (2009) Characterization of the PMT gene family in Cryptococcus neoformans. PLoS One 4:e6321. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Woloshuk CP, Foutz KR, Brewer JF, Bhatnagar D, Cleveland TE, Payne GA (1994) Molecular characterization of aflR, a regulatory locus for aflatoxin biosynthesis. Appl Environ Microbiol 60:2408–2414PubMedPubMedCentralGoogle Scholar
  54. Yu JH, Butchko RAE, Fernandes M, Keller NP, Leonard TJ, Adams TH (1996) Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus. Curr Genet 29:549–555CrossRefPubMedCentralGoogle Scholar
  55. Zhou H, Hu H, Zhang L, Li R, Ouyang H, Ming J, Jin C (2007) O-mannosyltransferase 1 in Aspergillus fumigatus (AfPmt1p) is crucial for cell wall integrity and conidium morphology, especially at an elevated temperature. Eukaryot Cell 6:2260–2268CrossRefPubMedCentralGoogle Scholar
  56. Zimmer D, Cornwall EH, Iandar A (1995) The S100 protein family: history, function, and expression. Brain Res Bull 37:417–429CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biological Science and TechnologyTokyo University of ScienceTokyoJapan
  2. 2.Division of Microbial BiotechnologyBiotechnology Center of Ho Chi Minh CityHo Chi Minh CityVietnam
  3. 3.Faculty of AgricultureSaga UniversitySagaJapan
  4. 4.Medical Mycology Research CenterChiba UniversityChibaJapan

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