Skip to main content

Advertisement

SpringerLink
Log in

Transcriptomic and Proteomic Profile of Aspergillus fumigatus on Exposure to Artemisinin

  • Published:
Mycopathologia Aims and scope Submit manuscript

Abstract

Artemisinin, an antimalarial drug, and its derivatives are reported to have antifungal activity against some fungi. We report its antifungal activity against Aspergillus fumigatus (A. fumigatus), a pathogenic filamentous fungus responsible for allergic and invasive aspergillosis in humans, and its synergistic effect in combination with itraconazole (ITC), an available antifungal drug. In order to identify its molecular targets, we further analyzed transcript and proteomic profiles of the fungus on exposure to the artemisinin. In transcriptomic analysis, a total of 745 genes were observed to be modulated on exposure to artemisinin, and some of them were confirmed by real-time polymerase chain reaction analysis. Proteomic profiles of A. fumigatus treated with artemisinin showed modulation of 175 proteins (66 upregulated and 109 downregulated) as compared to the control. Peptide mass fingerprinting led to the identification of 85 proteins—29 upregulated and 56 downregulated, 65 of which were unique proteins. Consistent with earlier reports of molecular mechanisms of artemisinin and that of other antifungal drugs, we believe that oxidative phosphorylation pathway (64 kDa mitochondrial NADH dehydrogenase), cell wall-associated proteins and enzymes (conidial hydrophobin B protein, cell wall phiA protein, extracellular thaumatin domain protein, 1,3-beta-glucanosyltransferase Gel2) and genes involved in ergosterol biosynthesis (ERG6 and coproporphyrinogen III oxidase, HEM13) are potential targets of artemisinin for further investigations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Brakhage AA. Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Current Drug Targets. 2005;6(8):875–86.

    Article  PubMed  CAS  Google Scholar 

  2. Pfaller MA, Pappas PG, Wingard JR. Invasive fungal pathogens: current epidemiological trends. Clin Infect Dis. 2006;43:S3–14.

    Article  CAS  Google Scholar 

  3. Ellis D. Amphotericin B: spectrum and resistance. J Antimicrob Chemother. 2002;9:7–10.

    Google Scholar 

  4. Kontoyiannis DP, Lewis RE. Antifungal drug resistance of pathogenic fungi. Lancet. 2002;359:1135–44.

    Article  PubMed  CAS  Google Scholar 

  5. Tan RX, Lu H, Wolfender JL, Yu TT, Zheng WF, Yang L, Gafner S, Hostettmann K. Mono- and sesquiterpenes and antifungal constituents from Artemisia species. Planta Med. 1999;65(1):64–7.

    Article  PubMed  CAS  Google Scholar 

  6. Galal AM, Ross SA, Jacob M, ElSohly MA. Antifungal activity of artemisinin derivatives. J Nat Prod. 2005;68(8):1274–6.

    Article  PubMed  CAS  Google Scholar 

  7. Dhingra V, Pakki SR, Narasu ML. Antimicrobial activity of artemisinin and its precursors. Curr Sci. 2000;78(6):709–13.

    CAS  Google Scholar 

  8. Pandey AV, Tekwani BL, Singh RL, Chauhan VS. Artemisinin, an endoperoxide antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in malarial parasite. J Biol Chem. 1999;274(27):19383–8.

    Article  PubMed  CAS  Google Scholar 

  9. Francis SE, Gluzman IY, Oksman A, Knickerbocker A, Mueller R, Bryant ML, Sherman DR, Russell DG, Goldberg DE. Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase. EMBO J. 1994;13(2):306–17.

    PubMed  CAS  Google Scholar 

  10. Salas F, Fichmann J, Lee GK, Scott MD, Rosenthal PJ. Functional expression of falcipain, a Plasmodium falciparum cysteine proteinase, supports its role as a malarial hemoglobinase. Infect Immun. 1995;63(6):2120–5.

    PubMed  CAS  Google Scholar 

  11. Gluzman IY, Francis SE, Oksman A, Smith CE, Duffin KL, Goldberg DE. Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway. J Clin Invest. 1994;93(4):1602–8.

    Article  PubMed  CAS  Google Scholar 

  12. Francis SE, Sullivan DJ Jr, Goldberg DE. Hemoglobin metabolism in the malaria parasite Plasmodium falciparum. Annu Rev Microbiol. 1997;51:97–123. Review.

    Article  PubMed  CAS  Google Scholar 

  13. Schwarzer E, Turrini F, Ulliers D, Giribaldi G, Ginsburg H, Arese P. Impairment of macrophage functions after ingestion of Plasmodium falciparum-infected erythrocytes or isolated malarial pigment. J Exp Med. 1992;176(4):1033–41.

    Article  PubMed  CAS  Google Scholar 

  14. Vander Jagt DL, Hunsaker LA, Campos NM. Characterization of a hemoglobin-degrading, low molecular weight protease from Plasmodium falciparum. Mol Biochem Parasitol. 1986;18(3):389–400.

    Article  PubMed  CAS  Google Scholar 

  15. Vander Jagt DL, Hunsaker LA, Campos NM, Scaletti JV. Localization and characterization of hemoglobin-degrading aspartic proteinases from the malarial parasite Plasmodium falciparum. Biochim Biophys Acta. 1992;1122(3):256–64.

    Article  PubMed  CAS  Google Scholar 

  16. Sherry BA, Alava G, Tracey KJ, Martiney J, Cerami A, Slater AF. Malaria-specific metabolite hemozoin mediates the release of several potent endogenous pyrogens (TNF, MIP-1 alpha, and MIP-1 beta) in vitro, and altered thermoregulation in vivo. J Inflamm. 1995;45(2):85–96.

    PubMed  CAS  Google Scholar 

  17. Eckstein-Ludwig U, Webb RJ, Van Goethem ID, East JM, Lee AG, Kimura M, O’Neill PM, Bray PG, Ward SA, Krishna S. Artemisinins target the SERCA of Plasmodium falciparum. Nature. 2003;424(6951):957–61.

    Article  PubMed  CAS  Google Scholar 

  18. Li W, Mo W, Shen D, Sun L, Wang J, Lu S, Gitschier JM, Zhou B. Yeast model uncovers dual roles of mitochondria in action of artemisinin. PLoS Genet. 2005;1(3):e36.

    Article  PubMed  Google Scholar 

  19. Wang J, Huang L, Li J, Fan Q, Long Y, Li Y, Zhou B. Artemisinin directly targets malarial mitochondria through its specific mitochondrial activation. PLoS One. 2010;5(3):e9582.

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  21. Arikan S, Lozano-Chiu M, Paetznick V, Nangia S, Rex JH. Microdilution susceptibility testing of amphotericin B, itraconazole, and voriconazole against clinical isolates of Aspergillus and Fusarium species. J Clin Microbiol. 1999;37(12):3946–51.

    PubMed  CAS  Google Scholar 

  22. Mosquera J, Sharp A, Moore CB, Warn PA, Denning DW. In vitro interaction of terbinafine with itraconazole, fluconazole, amphotericin B and 5-flucytosine against Aspergillus spp. J Antimicrob Chemother. 2002;50(2):189–94.

    Article  PubMed  CAS  Google Scholar 

  23. Vaquerizas JM, Dopazo J, Díaz-Uriarte R. DNMAD: web-based diagnosis and normalization for microarray data. Bioinformatics. 2004;20(18):3656–8.

    Article  PubMed  CAS  Google Scholar 

  24. Puckette MC, Tang Y, Mahalingam R. Transcriptomic changes induced by acute ozone in resistant and sensitive Medicago truncatula accessions. BMC Plant Biol. 2008;4(8):46.

    Article  Google Scholar 

  25. Semighini CP, Marins M, Goldman MHS, Goldman GH. Quantitative analysis of the relative transcript levels of ABC transporter Atr genes in Aspergillus nidulans by real-time reverse transcription-PCR assay. Appl Environ Microbiol. 2002;3:1351–7.

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  27. Görg A, Postel W, Günther S, Friedrich C. Horizontal two-dimensional electrophoresis with immobilized pH gradients using PhastSystem. Electrophoresis. 1988;9(1):57–9.

    Article  PubMed  Google Scholar 

  28. Chumbalkar VC, Subhashini C, Dhople VM, Sundaram CS, Jagannadham MV, Kumar KN, Srinivas PN, Mythili R, Rao MK, Kulkarni MJ, Hegde S, Hegde AS, Samual C, Santosh V, Singh L, Sirdeshmukh R. Differential protein expression in human gliomas and molecular insights. Proteomics. 2005;5(4):1167–77.

    Article  PubMed  CAS  Google Scholar 

  29. Zhang L, Zhang Y, Zhou Y, An S, Zhou Y, Cheng J. Response of gene expression in Saccharomyces cereviseae to amphotericin B and nystatin measured by microarrays. J Antimicrob Chemother. 2002;49(6):905–15.

    Article  PubMed  CAS  Google Scholar 

  30. Agarwal AK, Rogers PD, Baerson SR, Jacob MR, Barker KS, Cleary JD, Walker LA, Nagle DG, Clark AM. Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cereviseae. J Biol Chem. 2003;278(37):34998–5015.

    Article  PubMed  CAS  Google Scholar 

  31. Liu TT, Lee RE, Barker KS, Lee RE, Wei L, Homayouni R, Rogers PD. Genome-wide expression profiling of the response to azole, polyene, echinocandin, and pyrimidine antifungal agents in Candida albicans. Antimicrob Agents Chemother. 2005;49(6):2226–36.

    Article  PubMed  CAS  Google Scholar 

  32. da Silva Ferreira ME, Malavazi I, Savoldi M, Brakhage AA, Goldman MH, Kim HS, Nierman WC, Goldman GH. Transcriptome analysis of Aspergillus fumigatus exposed to voriconazole. Curr Genet. 2006;50(1):32–44.

    Article  PubMed  Google Scholar 

  33. Prieto JH, Koncarevic S, Park SK, Yates J 3rd, Becker K. Large-scale differential proteome analysis in Plasmodium falciparum under drug treatment. PLoS One. 2008;3(12):e4098.

    Article  PubMed  Google Scholar 

  34. Yu L, Zhang W, Wang L, Yang J, Liu T, Peng J, Leng W, Chen L, Li R, Jin Q. Transcriptional profiles of the response to ketoconazole and amphotericin B in Trichophyton rubrum. Antimicrob Agents Chemother. 2007;51(1):144–53.

    Article  PubMed  CAS  Google Scholar 

  35. Paris S, Debeaupuis JP, Crameri R, Carey M, Charles F, Prevost MC, Schmitt C, Philippe B, Latge JP. Conidial hydrophobins of Aspergillus fumigatus. Appl Environ Microbiol. 2003;69(3):1581–8.

    Article  PubMed  CAS  Google Scholar 

  36. Aimanianda V, Bayry J, Bozza S, Kniemeyer O, Perruccio K, Elluru SR, Clavaud C, Paris S, Brakhage AA, Kaveri SV, Romani L, Latgé JP. Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature. 2009;460(7259):1117–21.

    Article  PubMed  CAS  Google Scholar 

  37. Upadhyay SK, Mahajan L, Ramjee S, Singh Y, Basir SF, Madan T. Identification and characterization of a laminin-binding protein of Aspergillus fumigatus: extracellular thaumatin domain protein (AfCalAp). J Med Microbiol. 2009;58(Pt 6):714–22.

    Article  PubMed  CAS  Google Scholar 

  38. Gautam P, Sundaram CS, Madan T, Gade WN, Shah A, Sirdeshmukh R, Sarma PU. Identification of novel allergens of Aspergillus fumigatus using immunoproteomics approach. Clin Exp Allergy. 2007;37(8):1239–49.

    Article  PubMed  CAS  Google Scholar 

  39. Mouyna I, Morelle W, Vai M, Monod M, Léchenne B, Fontaine T, Beauvais A, Sarfati J, Prévost MC, Henry C, Latgé JP. Deletion of GEL2 encoding for a beta(1–3) glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus. Mol Microbiol. 2005;56(6):1675–88.

    Article  PubMed  CAS  Google Scholar 

  40. Bernard M, Latgé JP. Aspergillus fumigatus cell wall: composition and biosynthesis. Med Mycol. 2001;39(Suppl 1):9–17. Review.

    PubMed  CAS  Google Scholar 

  41. De Backer MD, Ilyina T, Ma XJ, Vandoninck S, Luyten WH, Vanden Bossche H. Genomic profiling of the response of Candida albicans to itraconazole treatment using a DNA microarray. Antimicrob Agents Chemother. 2001;45(6):1660–70.

    Article  PubMed  Google Scholar 

  42. Barker KS, Crisp S, Wiederhold N, Lewis RE, Bareither B, Eckstein J, Barbuch R, Bard M, Rogers PD. Genome-wide expression profiling reveals genes associated with amphotericin B and fluconazole resistance in experimentally induced antifungal resistant isolates of Candida albicans. J Antimicrob Chemother. 2004;54(2):376–85.

    Article  PubMed  CAS  Google Scholar 

  43. Alcazar-Fuoli L, Mellado E, Garcia-Effron G, Buitrago MJ, Lopez JF, Grimalt JO, Cuenca-Estrella JM, Rodriguez-Tudela JL. Aspergillus fumigatus C-5 sterol desaturases Erg3A and Erg3B: role in sterol biosynthesis and antifungal drug susceptibility. Antimicrob Agents Chemother. 2006;50(2):453–60.

    Article  PubMed  CAS  Google Scholar 

  44. Sarma PU, Kurup VP, Madan T. Immunodiagnosis of ABPA. Front Biosci. 2003;01:1187–98.

    Article  Google Scholar 

  45. Banerjee B, Kurup VP, Greenberger PA, Hoffman DR, Nair DS, Fink JN. Purification of a major allergen, Asp f 2 binding to IgE in allergic bronchopulmonary aspergillosis, from culture filtrate of Aspergillus fumigatus. J Allergy Clin Immunol. 1997;99:821–7.

    Article  PubMed  CAS  Google Scholar 

  46. Bhisutthibhan J, Pan XQ, Hossler PA, Walker DJ, Yowell CA, Carlton J, Dame JB, Meshnick SR. The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin. J Biol Chem. 1998;273(26):16192–8.

    Article  PubMed  CAS  Google Scholar 

  47. Ferreira ME, Colombo AL, Paulsen I, Ren Q, Wortman J, Huang J, Goldman MH, Goldman GH. The ergosterol biosynthesis pathway, transporter genes, and azole resistance in Aspergillus fumigatus. Med Mycol. 2005;43(Suppl 1):S313–9.

    Article  PubMed  Google Scholar 

  48. Slaven JW, Anderson MJ, Sanglard D, Dixon GK, Bille J, Roberts IS, Denning DW. Increased expression of a novel Aspergillus fumigatus ABC transporter gene, atrF, in the presence of itraconazole in an itraconazole resistant clinical isolate. Fungal Genet Biol. 2002;36:199–206.

    Article  PubMed  CAS  Google Scholar 

  49. Nascimento AM, Goldman GH, Park S, Marras SA, Delmas G, Oza U, Lolans K, Dudley MN, Mann PA, Perlin DS. Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole. Antimicrob Agents Chemother. 2003;47(5):1719–26.

    Article  PubMed  CAS  Google Scholar 

  50. Coleman JJ, Mylonakis E. Efflux in fungi: la pièce de résistance. PLoS Pathog. 2009;5(6):e1000486.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We are grateful to Council of Scientific and Industrial Research and Department of Science and Technology, Government of India, for the financial support. We are grateful to Pathogen Functional Genomics Resource Center (PFGRC) at J. Craig Venter Institute (JCVI, Rockville, Maryland, USA) for providing A. fumigatus microarray slides. We are also thankful to The Centre for Genomic Application (TCGA) for providing the microarray facility. We acknowledge Dr. D. W. Denning, School of Medicine, University of Manchester, Manchester, UK, for providing the Af293 strain. Dr. Poonam Gautam was recipient of Research Associate Fellowship of Department of Science and Technology, Government of India, and Dr. Santosh Kumar Upadhyay was recipient of Senior Research Fellowship of Council of Scientific and Industrial Research, Government of India.

Author information

Authors and Affiliations

  1. Institute for Genomics and Integrative Biology, Delhi, India

    Poonam Gautam, Santosh Kumar Upadhyay, Wazid Hassan, Taruna Madan, Yogendra Singh & Puranam Usha Sarma

  2. Centre for Cellular and Molecular Biology, Hyderabad, India

    Poonam Gautam, Ravi Sirdeshmukh & Curam Sreenivasacharlu Sundaram

  3. Innate Immunity, National Institute for Research in Reproductive Health (NIRRH), Parel, Mumbai, 400012, India

    Taruna Madan

  4. Department of Biotechnology, University of Pune, Pune, India

    Poonam Gautam & Wasudev Namdeo Gade

  5. Department of Biosciences, Jamia Milia Islamia, Delhi, India

    Santosh Kumar Upadhyay & Seemi Farhat Basir

  6. Department of Plant Pathology, Indian Agricultural Research Institute, Delhi, India

    Puranam Usha Sarma

Authors
  1. Poonam Gautam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Santosh Kumar Upadhyay
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wazid Hassan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Taruna Madan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ravi Sirdeshmukh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Curam Sreenivasacharlu Sundaram
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wasudev Namdeo Gade
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Seemi Farhat Basir
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yogendra Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Puranam Usha Sarma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Taruna Madan.

Additional information

Poonam Gautam and Santosh Kumar Upadhyay contributed equally to this work.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gautam, P., Upadhyay, S.K., Hassan, W. et al. Transcriptomic and Proteomic Profile of Aspergillus fumigatus on Exposure to Artemisinin. Mycopathologia 172, 331–346 (2011). https://doi.org/10.1007/s11046-011-9445-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11046-011-9445-3

Keywords

Search

Navigation