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Nano-LC-Q-TOF Analysis of Proteome Revealed Germination of Aspergillus flavus Conidia is Accompanied by MAPK Signalling and Cell Wall Modulation


Aspergillus flavus is the second most leading cause of aspergillosis. The ability of A. flavus to adapt within the host environment is crtical for its colonization. Onset of germination of conidia is one of the crucial events; thus, in order to gain insight into A. flavus molecular adaptation while germination, protein profile of A. flavus was obtained. Approximately 82 % of conidia showed germination at 7 h; thus, samples were collected followed by protein extraction and subjected to nLC-Q-TOF mass spectrometer. Q-TOF data were analysed using Protein Lynx Global Services (PLGS 2.2.5) software. A total of 416 proteins were identified from UniProt Aspergillus species database. Orthologues of A. flavus was observed in A. fumigatus, A. niger, A. terreus, A. oryzae, etc. Proteins were further analysed in NCBI database, which showed that 27 proteins of A. flavus are not reported in UniProt and NCBI database. Functional characterization of proteins resulted majorly to cell wall synthesis and degradation, metabolisms (carbohydrate and amino acid metabolism), protein synthesis and degradation. Proteins/enzymes associated with aflatoxin biosynthesis were observed. We also observed Dicer-like proteins 1, 2 and autophagy-related proteins 2, 9, 18, 13, 11, 22. Expression of protein/enzymes associated with MAPK signalling pathway suggests their role during the germination process. Overall, the data present a catalogue of proteins/enzymes involved in the germination of A. flavus conidia and could also be applied to other Aspergillus species.

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  1. 1.

    Thakur R, Anand R, Tiwari S, Singh AP, Tiwary BN, Shankar J. Cytokines induce effector T-helper cells during invasive aspergillosis; what we have learned about T-helper cells? Front Microbiol. 2015;6:429.

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Guarro J, Xavier MO, Severo LC. Differences and similarities amongst pathogenic Aspergillus species. Aspergillosis: from diagnosis to prevention. New York: Springer; 2009. p. 7–32.

    Chapter  Google Scholar 

  3. 3.

    Chowdhary A, Sharma C, Kathuria S, Hagen F, Meis JF. Prevalence and mechanism of triazole resistance in Aspergillus fumigatus in a referral chest hospital in Delhi, India and an update of the situation in Asia. Front Microbiol. 2015;6:428.

    Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Ramírez-Camejo LA, Zuluaga-Montero A, Lázaro-Escudero M, Hernández-Kendall V, Bayman P. Phylogeography of the cosmopolitan fungus Aspergillus flavus: is everything everywhere? Fungal Biol. 2012;116(3):452–63.

    Article  PubMed  Google Scholar 

  5. 5.

    Bosetti C, Levi F, Boffetta P, Lucchini F, Negri E, La Vecchia C. Trends in mortality from hepatocellular carcinoma in Europe, 1980–2004. Hepatology. 2008;48(1):137–45.

    Article  PubMed  Google Scholar 

  6. 6.

    Hedayati M, Pasqualotto A, Warn P, Bowyer P, Denning D. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology. 2007;153(6):1677–92.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Yu J, Payne GA, Nierman WC, Machida M, Bennett JW, Campbell BC, et al. Aspergillus flavus genomics as a tool for studying the mechanism of aflatoxin formation. Food Addit Contam. 2008;25(9):1152–7.

    CAS  Article  Google Scholar 

  8. 8.

    Bhatnagar D, Cary JW, Ehrlich K, Yu J, Cleveland TE. Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development. Mycopathologia. 2006;162(3):155–66.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Van Leeuwen M, Van Doorn T, Golovina E, Stark J, Dijksterhuis J. Water-and air-distributed conidia differ in sterol content and cytoplasmic microviscosity. Appl Environ Microbiol. 2010;76(1):366–9.

    Article  PubMed  Google Scholar 

  10. 10.

    Osherov N, May GS. The molecular mechanisms of conidial germination. FEMS Microbiol Lett. 2001;199(2):153–60.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Harris SD. Cell polarity in filamentous fungi: shaping the mold. Int Rev Cytol. 2006;251:41–77.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Pechanova O, Pechan T, Rodriguez JM, PaulWilliams W, Brown AE. A two-dimensional proteome map of the aflatoxigenic fungus Aspergillus flavus. Proteomics. 2013;13(9):1513–8.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Medina ML, Haynes PA, Breci L, Francisco WA. Analysis of secreted proteins from Aspergillus flavus. Proteomics. 2005;5(12):3153–61.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Georgianna DR, Hawkridge AM, Muddiman DC, Payne GA. Temperature-dependent regulation of proteins in Aspergillus flavus: whole organism stable isotope labeling by amino acids. J Proteome Res. 2008;7(7):2973–9.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Suh M-J, Fedorova ND, Cagas SE, Hastings S, Fleischmann RD, Peterson SN, et al. Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus fumigatus conidial proteome. Proteome Sci. 2012;10(1):1.

    Article  Google Scholar 

  16. 16.

    Kubitschek-Barreira PH, Curty N, Neves GW, Gil C, Lopes-Bezerra LM. Differential proteomic analysis of Aspergillus fumigatus morphotypes reveals putative drug targets. J Proteom. 2013;78:522–34.

    CAS  Article  Google Scholar 

  17. 17.

    Leng W, Liu T, Li R, Yang J, Wei C, Zhang W, et al. Proteomic profile of dormant Trichophyton Rubrum conidia. BMC Genom. 2008;9(1):303.

    Article  Google Scholar 

  18. 18.

    Van Leeuwen M, Krijgsheld P, Bleichrodt R, Menke H, Stam H, Stark J, et al. Germination of conidia of Aspergillus niger is accompanied by major changes in RNA profiles. Stud Mycol. 2013;74:59–70.

    Article  PubMed  Google Scholar 

  19. 19.

    Lamarre C, Sokol S, Debeaupuis J-P, Henry C, Lacroix C, Glaser P, et al. Transcriptomic analysis of the exit from dormancy of Aspergillus fumigatus conidia. BMC Genom. 2008;9(1):417.

    Article  Google Scholar 

  20. 20.

    Ullmann AJ, Cornely OA. Antifungal prophylaxis for invasive mycoses in high risk patients. Curr Opin Infect Dis. 2006;19(6):571–6.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Valiante V, Macheleidt J, Föge M, Brakhage AA. The Aspergillus fumigatus cell wall integrity signaling pathway: drug target, compensatory pathways, and virulence. Front Microbiol. 2015;6:325.

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Gautam P, Shankar J, Madan T, Sirdeshmukh R, Sundaram CS, Gade WN, et al. Proteomic and transcriptomic analysis of Aspergillus fumigatus on exposure to amphotericin B. Antimicrob Agents Chemother. 2008;52(12):4220–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Gautam P, Mushahary D, Hassan W, Upadhyay SK, Madan T, Sirdeshmukh R, et al. In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole. Med Mycol. 2016;54(5):524–36.

    Article  PubMed  Google Scholar 

  24. 24.

    Bai Y, Wang S, Zhong H, Yang Q, Zhang F, Zhuang Z, et al. Integrative analyses reveal transcriptome-proteome correlation in biological pathways and secondary metabolism clusters in A. flavus in response to temperature. Sci Rep. 2015;29(5):14582. doi:10.1038/srep14582.

    Article  Google Scholar 

  25. 25.

    Anand R, Shankar J, Singh AP, Tiwary BN. Cytokine milieu in renal cavities of immunocompetent mice in response to intravenous challenge of Aspergillus flavus leading to aspergillosis. Cytokine. 2013;61(1):63–70.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Patel TK, Anand R, Singh AP, Shankar J, Tiwary BN. Evaluation of aflatoxin B1 biosynthesis in A. flavus isolates from central india and identification of atoxigenic isolates. Biotechnol Bioprocess Eng. 2014;19(6):1105–13.

    CAS  Article  Google Scholar 

  27. 27.

    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1–2):248–54.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Du L-Y, Zhao M, Xu J, Qian D-W, Jiang S, Shang E-X, et al. Identification of the metabolites of myricitrin produced by human intestinal bacteria in vitro using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. Expert Opin Drug Metab Toxicol. 2014;10(7):921–31.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Farrell A, Mittermayr S, Morrissey B, Mc Loughlin N, Navas Iglesias N, Marison IW, et al. Quantitative host cell protein analysis using two dimensional data independent LC–MSE. Anal Chem. 2015;87(18):9186–93.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Von Mering C, Jensen LJ, Snel B, Hooper SD, Krupp M, Foglierini M, et al. STRING: known and predicted protein–protein associations, integrated and transferred across organisms. Nucleic Acids Res. 2005;33(suppl 1):D433–7.

    Google Scholar 

  32. 32.

    Priebe S, Kreisel C, Horn F, Guthke R, Linde J. FungiFun2: a comprehensive online resource for systematic analysis of gene lists from fungal species. Bioinformatics. 2015;31(3):445–6.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Bernard M, Latgé J-P. Aspergillus fumigatus cell wall: composition and biosynthesis. Med Mycol. 2001;39(1):9–17.

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Henry C, Latgé J-P, Beauvais A. α1, 3 glucans are dispensable in Aspergillus fumigatus. Eukaryot Cell. 2012;11(1):26–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Firon A, Beauvais A, Latgé J-P, Couvé E, Grosjean-Cournoyer M-C, d’Enfert C. Characterization of essential genes by parasexual genetics in the human fungal pathogen Aspergillus fumigatus: impact of genomic rearrangements associated with electroporation of DNA. Genetics. 2002;161(3):1077–87.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Dichtl K, Samantaray S, Aimanianda V, Zhu Z, Prévost MC, Latgé JP, et al. Aspergillus fumigatus devoid of cell wall β-1, 3-glucan is viable, massively sheds galactomannan and is killed by septum formation inhibitors. Mol Microbiol. 2015;95(3):458–71.

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Jiménez-Ortigosa C, Aimanianda V, Muszkieta L, Mouyna I, Alsteens D, Pire S, et al. Chitin synthases with a myosin motor-like domain control the resistance of Aspergillus fumigatus to echinocandins. Antimicrob Agents Chemother. 2012;56(12):6121–31.

    Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Bayram Ö, Bayram ÖS, Ahmed YL, J-i Maruyama, Valerius O, Rizzoli SO, et al. The Aspergillus nidulans MAPK module AnSte11-Ste50-Ste7-Fus3 controls development and secondary metabolism. PLoS Genet. 2012;8(7):e1002816.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Dirr F, Echtenacher B, Heesemann J, Hoffmann P, Ebel F, Wagener J. AfMkk2 is required for cell wall integrity signaling, adhesion, and full virulence of the human pathogen Aspergillus fumigatus. Int J Med Microbiol. 2010;300(7):496–502.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Valiante V, Jain R, Heinekamp T, Brakhage AA. The MpkA MAP kinase module regulates cell wall integrity signaling and pyomelanin formation in Aspergillus fumigatus. Fungal Genet Biol. 2009;46(12):909–18.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Shankar J. An overview of toxins in Aspergillus associated with pathogenesis. Int J Life Sci Biotechnol Pharma Res. 2013;2(2):16–31.

    Google Scholar 

  42. 42.

    Wiederhold NP. Paradoxical echinocandin activity: a limited in vitro phenomenon? Med Mycol. 2009;47(sup1):S369–75.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Fortwendel JR, Juvvadi PR, Perfect BZ, Rogg LE, Perfect JR, Steinbach WJ. Transcriptional regulation of chitin synthases by calcineurin controls paradoxical growth of Aspergillus fumigatus in response to caspofungin. Antimicrob Agents Chemother. 2010;54(4):1555–63.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Champer J, Ito JI, Clemons KV, Stevens DA, Kalkum M. Proteomic analysis of pathogenic fungi reveals highly expressed conserved cell wall proteins. J Fungi. 2016;2(1):6.

    Article  Google Scholar 

  45. 45.

    Garciá-Ortega L, Lacadena J, Villalba M, Rodríguez R, Crespo JF, Rodríguez J, et al. Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding. FEBS J. 2005;272(10):2536–44.

    Article  PubMed  Google Scholar 

  46. 46.

    Tiwari S, Thakur R, Shankar J. Role of heat-shock proteins in cellular function and in the biology of fungi. Biotechnol Res Int. 2015;2015:132635. doi:10.1155/2015/132635.

  47. 47.

    Lamoth F, Juvvadi PR, Fortwendel JR, Steinbach WJ. Heat shock protein 90 is required for conidiation and cell wall integrity in Aspergillus fumigatus. Eukaryot Cell. 2012;11(11):1324–32.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Juvvadi PR, Lamoth F, Steinbach WJ. Calcineurin as a multifunctional regulator: unraveling novel functions in fungal stress responses, hyphal growth, drug resistance, and pathogenesis. Fungal Biol Rev. 2014;28(2):56–69.

    Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Pollack JK, Harris SD, Marten MR. Autophagy in filamentous fungi. Fungal Genet Biol. 2009;46(1):1–8.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Dang Y, Yang Q, Xue Z, Liu Y. RNA interference in fungi: pathways, functions, and applications. Eukaryot Cell. 2011;10(9):1148–55.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Liu X, Walsh CT. Cyclopiazonic acid biosynthesis in Aspergillus sp.: characterization of a reductase-like R* domain in cyclopiazonate synthetase that forms and releases cyclo-acetoacetyl-L-tryptophan. Biochemistry. 2009;48(36):8746–57.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Ramesh MV, Kolattukudy PE. Disruption of the serine proteinase gene (sep) in Aspergillus flavus leads to a compensatory increase in the expression of a metalloproteinase gene (mep20). J Bacteriol. 1996;178(13):3899–907.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Tsitsigiannis DI, Bok J-W, Andes D, Nielsen KF, Frisvad JC, Keller NP. Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin production and virulence. Infect Immun. 2005;73(8):4548–59.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Dias-Lopes C, Neshich IA, Neshich G, Ortega JM, Granier C, Chávez-Olortegui C, et al. Identification of new sphingomyelinases D in pathogenic fungi and other pathogenic organisms. PLoS ONE. 2013;8(11):e79240.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Authors are thankful to Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, for providing facilities and Ph.D. fellowship to ST and RT.

Authors Contributions

ST and JS conceived and designed the experiments. ST performed the experiments. ST, RT and JS analysed the data. GG and JS contributed reagents/materials/analysis tools. ST and JS contributed to writing the manuscript.

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Corresponding author

Correspondence to Jata Shankar.

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The authors declare that they have no conflit of interests.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Table S1 (A)

List of 416 proteins of Aspergillus flavus that are expressed at germinating stage (7 h). (XLSX 131 kb)

Supplementary Table S1 (B)

List of proteins identified in Aspergillus flavus grown on Sabouraud dextrose media for 7 h (germinating conidial stage). Proteins were separated and identified via nL-Q-TOF mass spectroscopy and are clustered according to biological process as defined by Gene Ontology from UniProt database. (XLSX 261 kb)

Supplementary Table S2

Details of selected protein of carbohydrate metabolism involved in various biological, molecular and cellular processes. The data represent the significance in relation to p values and the protein is represented by Gene Ontology IDs obtained from FungiFun 2.2.8 BETA software. (PDF 254 kb)

Supplementary material 4 (DOCX 19 kb)

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Tiwari, S., Thakur, R., Goel, G. et al. Nano-LC-Q-TOF Analysis of Proteome Revealed Germination of Aspergillus flavus Conidia is Accompanied by MAPK Signalling and Cell Wall Modulation. Mycopathologia 181, 769–786 (2016).

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  • Aspergillus flavus
  • Germinating conidia
  • nLC-Q-TOF
  • Proteome
  • Cell wall
  • Anti-Aspergillus targets