Abstract
Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests.
Key points
• CSPs and exoproteins could differentiate A. flavus and A. fumigatus.
• A. fumigatus conidial surface harbored more antigenic proteins than A. flavus.
• Identified species-specific signature proteins of A. flavus and A. fumigatus.
Graphical Abstract
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers:
• PXD022824 for the conidial surface-associated protein identification data
• PXD012982 for the exoproteome identification data.
References
Aguirre J, Hansberg W, Navarro R (2006) Fungal responses to reactive oxygen species. Med Mycol 44:S101–S107. https://doi.org/10.1080/13693780600900080
Aimanianda V, Bayry J, Bozza S, Kniemeyer O, Perruccio K, Elluru SR, Clavaud C, Paris S, Brakhage AA, Kaveri SV, Romani L, Latgé JP (2009) Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature 460(7259):1117–1121. https://doi.org/10.1038/nature08264
Albrandt K, Orida NK, Liu FT (1987) An IgE-binding protein with a distinctive repetitive sequence and homology with an IgG receptor. Proc Natl Acad Sci U S A 84:6859–6863. https://doi.org/10.1073/pnas.84.19.6859
Amich J, Vicentefranqueira R, Leal F, Calera JA (2010) Aspergillus fumigatus survival in alkaline and extreme zinc-limiting environments relies on the induction of a zinc homeostasis system encoded by the zrfC and aspf2 genes. Eukaryot Cell 9(3):424–437. https://doi.org/10.1128/EC.00348-09
Balloy V, Sallenave JM, Wu Y, Touqui L, Latgé JP, Si-Tahar M, Chignard M (2008) Aspergillus fumigatus-induced interleukin-8 synthesis by respiratory epithelial cells is controlled by the phosphatidylinositol 3-kinase, p38 MAPK, and ERK1/2 pathways and not by the toll-like receptor-MyD88 pathway. J Biol Chem 283(45):30513–30521. https://doi.org/10.1074/jbc.M803149200
Banerjee B, Greenberger PA, Fink JN, Kurup VP (1998) Immunological characterization of Asp f 2, a major allergen from Aspergillus fumigatus associated with allergic bronchopulmonary aspergillosis. Infect Immun 66(11):5175–5182. https://doi.org/10.1128/IAI.66.11.5175-5182.1998
Bayry J, Aimanianda V, Guijarro JI, Sunde M, Latgé JP (2012) Hydrophobins--unique fungal proteins. PLoS Pathog 8(5):e1002700 https://doi.org/10.1371/journal.ppat.1002700
Bayry J, Beaussart A, Dufrêne YF, Sharma M, Bansal K, Kniemeyer O, Aimanianda V, Brakhage AA, Kaveri SV, Kwon-Chung KJ, Latgé JP, Beauvais A (2014) Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response. Infect Immun 82(8):3141–3153. https://doi.org/10.1128/IAI.01726-14
Boynton AL, Whitfield JF, MacManus JP (1980) Calmodulin stimulates DNA synthesis by rat liver cells. Biochem Biophys Res Commun 95(2):745–749. https://doi.org/10.1016/0006-291x(80)90849-9
Breitenbach M, Weber M, Rinnerthaler M, Karl T, Breitenbach-Koller L (2015) Oxidative stress in fungi: its function in signal transduction, interaction with plant hosts, and lignocellulose degradation. Biomolecules 5(2):318–342. https://doi.org/10.3390/biom5020318
Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC (2012) Hidden killers: human fungal infections. Sci Transl Med 4(165):165rv13. https://doi.org/10.1126/scitranslmed.3004404
Calera JA, Paris S, Monod M, Hamilton AJ, Debeaupuis JP, Diaquin M, López-Medrano R, Leal F, Latgé JP (1997) Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus. Infect Immun 65(11):4718–4724. https://doi.org/10.1128/IAI.65.11.4718-4724.1997
Carrion Sde J, Leal SM Jr, Ghannoum MA, Aimanianda V, Latgé JP, Pearlman E (2013) The RodA hydrophobin on Aspergillus fumigatus spores masks dectin-1- and dectin-2-dependent responses and enhances fungal survival in vivo. J Immunol 191(5):2581–2588. https://doi.org/10.4049/jimmunol.1300748
Chabane S, Sarfati J, Ibrahim-Granet O, Du C, Schmidt C, Mouyna I, Prevost MC, Calderone R, Latgé JP (2006) Glycosylphosphatidylinositol-anchored Ecm33p influences conidial cell wall biosynthesis in Aspergillus fumigatus. Appl Environ Microbiol 72(5):3259–3267. https://doi.org/10.1128/AEM.72.5.3259-3267.2006
Champer J, Diaz-Arevalo D, Champer M, Hong TB, Wong M, Shannahoff M, Ito JI, Clemons KV, Stevens DA, Kalkum M (2012) Protein targets for broad-spectrum mycosis vaccines: quantitative proteomic analysis of Aspergillus and Coccidioides and comparisons with other fungal pathogens. Ann N Y Acad Sci 1273:44–51. https://doi.org/10.1111/j.1749-6632.2012.06761.x
Chaudhuri R, Ansari FA, Raghunandanan MV, Ramachandran S (2011) FungalRV: adhesin prediction and immunoinformatics portal for human fungal pathogens. BMC Genomics 12:192. https://doi.org/10.1186/1471-2164-12-192
Chowdhary A, Sharma C, Meis JF (2017) Azole-resistant aspergillosis: epidemiology, molecular mechanisms, and treatment. J Infect Dis 216(suppl_3):S436–S444. https://doi.org/10.1093/infdis/jix210
Crameri R, Glaser AG, Vilhelmsson M, Zeller S, Rhyner C (2010) Overview of Aspergillus allergens. In: Pasqualotto AC (ed) Aspergillosis: from diagnosis to prevention. Springer, London, pp 655–669
De Wit PJ, Mehrabi R, Van den Burg HA, Stergiopoulos I (2009) Fungal effector proteins: past, present and future. Mol Plant Pathol 10(6):735–747. https://doi.org/10.1111/j.1364-3703.2009.00591.x
Fellbrich G, Romanski A, Varet A, Blume B, Brunner F, Engelhardt S, Felix G, Kemmerling B, Krzymowska M, Nürnberger T (2002) NPP1, a Phytophthora-associated trigger of plant defense in parsley and Arabidopsis. Plant J 32(3):375–390. https://doi.org/10.1046/j.1365-313x.2002.01454.x
Gautam P, Sundaram CS, Madan T, Gade WN, Shah A, Sirdeshmukh R, Sarma PU (2007) Identification of novel allergens of Aspergillus fumigatus using immunoproteomics approach. Clin Exp Allergy 37(8):1239–1249. https://doi.org/10.1111/j.1365-2222.2007.02765.x
Gijzen M, Nürnberger T (2006) Nep1-like proteins from plant pathogens: recruitment and diversification of the NPP1 domain across taxa. Phytochemistry 67(16):1800–1807. https://doi.org/10.1016/j.phytochem.2005.12.008
Girard V, Dieryckx C, Job C, Job D (2013) Secretomes: the fungal strike force. Proteomics 13(3-4):597–608. https://doi.org/10.1002/pmic.201200282
Glaser AG, Kirsch AI, Zeller S, Menz G, Rhyner C, Crameri R (2009) Molecular and immunological characterization of Asp f 34, a novel major cell wall allergen of Aspergillus fumigatus. Allergy 64(8):1144–1151. https://doi.org/10.1111/j.1398-9995.2009.02029.x
Goberdhan NJ, Dawson RA, Freedlander E, Mac Neil S (1993) A calmodulin-like protein as an extracellular mitogen for the keratinocyte. Br J Dermatol 129(6):678–688. https://doi.org/10.1111/j.1365-2133.1993.tb03331.x
Haiech J, Audran E, Fève M, Ranjeva R, Kilhoffer MC (2011) Revisiting intracellular calcium signaling semantics. Biochimie 93(12):2029–2037. https://doi.org/10.1016/j.biochi.2011.05.003
Hedayati MT, Pasqualotto AC, Warn PA, Bowyer P, Denning DW (2007) Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology 153:1677–1692. https://doi.org/10.1099/mic.0.2007/007641-01
Heddergott C, Calvo AM, Latgé JP (2014) The volatome of Aspergillus fumigatus. Eukaryot Cell 13(8):1014–1025. https://doi.org/10.1128/EC.00074-14
Hillmann F, Bagramyan K, Strassburger M, Heinekamp T, Hong TB, Bzymek KP, Williams JC, Brakhage AA, Kalkum M (2016) The crystal structure of peroxiredoxin Asp f3 provides mechanistic insight into oxidative stress resistance and virulence of Aspergillus fumigatus. Sci Rep 6:33396. https://doi.org/10.1038/srep33396
Houser J, Komarek J, Kostlanova N, Cioci G, Varrot A, Kerr SC, Lahmann M, Balloy V, Fahy JV, Chignard M, Imberty A, Wimmerova M (2013) A soluble fucose-specific lectin from Aspergillus fumigatus conidia--structure, specificity and possible role in fungal pathogenicity. PLoS One 8(12):e83077. https://doi.org/10.1371/journal.pone.0083077
Jaton-Ogay K, Paris S, Huerre M, Quadroni M, Falchetto R, Togni G, Latgé JP, Monod M (1994) Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus. Mol Microbiol 14(5):917–928. https://doi.org/10.1111/j.1365-2958.1994.tb01327.x
Kandhavelu J, Demonte NL, Namperumalsamy VP, Prajna L, Thangavel C, Jayapal JM, Kuppamuthu D (2017) Aspergillus flavus induced alterations in tear protein profile reveal pathogen-induced host response to fungal infection. J Proteome 152:13–21. https://doi.org/10.1016/j.jprot.2016.10.009
Kao R, Martínez-Ruiz A, Martínez del Pozo A, Crameri R, Davies J (2001) Mitogillin and related fungal ribotoxins. Methods Enzymol 341:324–335. https://doi.org/10.1016/s0076-6879(01)41161-x
Karkowska-Kuleta J, Kozik A (2015) Cell wall proteome of pathogenic fungi. Acta Biochim Pol 62(3):339–351. https://doi.org/10.18388/abp.2015_1032
Keller A, Ai N, Kolker E, Aebersold R (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74(20):5383–5392. https://doi.org/10.1021/ac025747h
Kolattukudy PE, Lee JD, Rogers LM, Zimmerman P, Ceselski S, Fox B, Stein B, Copelan EA (1993) Evidence for possible involvement of an elastolytic serine protease in aspergillosis. Infect Immun 61(6):2357–2368. https://doi.org/10.1128/IAI.61.6.2357-2368.1993
Kumar A, Ahmed R, Singh PK, Shukla PK (2011) Identification of virulence factors and diagnostic markers using immunosecretome of Aspergillus fumigatus. J Proteome 74(7):1104–1112. https://doi.org/10.1016/j.jprot.2011.04.004
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Lalitha P, Prajna NV, Manoharan G, Srinivasan M, Mascarenhas J, Das M, D'Silva SS, Porco TC, Keenan JD (2015) Trends in bacterial and fungal keratitis in South India, 2002-2012. Br J Ophthalmol 99(2):192–194. https://doi.org/10.1136/bjophthalmol-2014-305000
Latgé JP, Chamilos G (2019) Aspergillus fumigatus and aspergillosis in 2019. Clin Microbiol Rev 33(1):e00140. https://doi.org/10.1128/CMR.00140-18
Lee JD, Kolattukudy PE (1995) Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung. Infect Immun 63(10):3796–3803. https://doi.org/10.1128/IAI.63.10.3796-3803.1995
Liu Q, Li JG, Ying SH, Wang JJ, Sun WL, Tian CG, Feng MG (2015) Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen. Environ Microbiol 17(4):1444–1462. https://doi.org/10.1111/1462-2920.12634
Luhtala N, Parker R (2010) T2 family ribonucleases: ancient enzymes with diverse roles. Trends Biochem Sci 35(5):253–259. https://doi.org/10.1016/j.tibs.2010.02.002
Mir AA, Park SY, Abu Sadat M, Kim S, Choi J, Jeon J, Lee YH (2015) Systematic characterization of the peroxidase gene family provides new insights into fungal pathogenicity in Magnaporthe oryzae. Sci Rep 5:11831. https://doi.org/10.1038/srep11831
Miyara I, Shafran H, Davidzon M, Sherman A, Prusky D (2010) pH regulation of ammonia secretion by Colletotrichum gloeosporioides and its effect on appressorium formation and pathogenicity. Mol Plant-Microbe Interact 23(3):304–316. https://doi.org/10.1094/MPMI-23-3-0304
Moore GE, Gerner RE, Franklin HA (1967) Culture of normal human leukocytes. JAMA 199:519–524
Mouyna I, Hartland RP, Fontaine T, Diaquin M, Simenel C, Delepierre M, Henrissat B, Latgé JP (1998) A 1,3-ß-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus is a homologue of the yeast Bgl2p. Microbiology (Reading) 144(Pt 11):3171–3180. https://doi.org/10.1099/00221287-144-11-3171
Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75(17):4646–4658. https://doi.org/10.1021/ac0341261
Paris S, Wysong D, Debeaupuis JP, Shibuya K, Philippe B, Diamond RD, Latgé JP (2003) Catalases of Aspergillus fumigatus. Infect Immun 71(6):3551–3562. https://doi.org/10.1128/iai.71.6.3551-3562.2003
Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, Inuganti A, Griss J, Mayer G, Eisenacher M, Pérez E, Uszkoreit J, Pfeuffer J, Sachsenberg T, Yilmaz S, Tiwary S, Cox J, Audain E, Walzer M et al (2019) The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res 47(D1):D442–D450. https://doi.org/10.1093/nar/gky1106
Ramirez-Garcia A, Pellon A, Buldain I, Antoran A, Arbizu-Delgado A, Guruceaga X, Rementeria A, Hernando FL (2018) Proteomics as a tool to identify new targets against Aspergillus and Scedosporium in the context of cystic fibrosis. Mycopathologia 183(1):273–289. https://doi.org/10.1007/s11046-017-0139-3
Reichard U, Büttner S, Eiffert H, Staib F, Rüchel R (1990) Purification and characterisation of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue. J Med Microbiol 33(4):243–251. https://doi.org/10.1099/00222615-33-4-243
Reichard U, Monod M, Odds F, Rüchel R (1997) Virulence of an aspergillopepsin-deficient mutant of Aspergillus fumigatus and evidence for another aspartic proteinase linked to the fungal cell wall. J Med Vet Mycol 35(3):189–196. https://doi.org/10.1080/02681219780001131
Romano J, Nimrod G, Ben-Tal N, Shadkchan Y, Baruch K, Sharon H, Osherov N (2006) Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence. Microbiology (Reading) 152(Pt 7):1919–1928. https://doi.org/10.1099/mic.0.28936-0
Rudramurthy SM, Paul RA, Chakrabarti A, Mouton JW, Meis JF (2019) Invasive aspergillosis by Aspergillus flavus: epidemiology, diagnosis, antifungal resistance, and management. J Fungi (Basel) 5(3):55. https://doi.org/10.3390/jof5030055
Salazar F, Ghaemmaghami AM (2013) Allergen recognition by innate immune cells: critical role of dendritic and epithelial cells. Front Immunol 4:356. https://doi.org/10.3389/fimmu.2013.00356
Savino S, Fraaije MW (2021) The vast repertoire of carbohydrate oxidases: an overview. Biotechnol Adv 51:107634. https://doi.org/10.1016/j.biotechadv.2020.107634
Schwienbacher M, Weig M, Thies S, Regula JT, Heesemann J, Ebel F (2005) Analysis of the major proteins secreted by the human opportunistic pathogen Aspergillus fumigatus under in vitro conditions. Med Mycol 43(7):623–630. https://doi.org/10.1080/13693780500089216
Singh B, Oellerich M, Kumar R, Kumar M, Bhadoria DP, Reichard U, Gupta VK, Sharma GL, Asif AR (2010) Immuno-reactive molecules identified from the secreted proteome of Aspergillus fumigatus. J Proteome Res 9(11):5517–5529. https://doi.org/10.1021/pr100604x
Sperschneider J, Gardiner DM, Dodds PN, Tini F, Covarelli L, Singh KB, Manners JM, Taylor JM (2016) EffectorP: predicting fungal effector proteins from secretomes using machine learning. New Phytol 210(2):743–761. https://doi.org/10.1111/nph.13794
Srinivasa K, Kim NR, Kim J, Kim M, Bae JY, Jeong W, Kim W, Choi W (2012) Characterization of a putative thioredoxin peroxidase prx1 of Candida albicans. Mol Cells 33(3):301–307. https://doi.org/10.1007/s10059-012-2260-y
Stergiopoulos I, de Wit PJ (2009) Fungal effector proteins. Annu Rev Phytopathol 47:233–263. https://doi.org/10.1146/annurev.phyto.112408.132637
Svanström Å, Boveri S, Boström E, Melin P (2013) The lactic acid bacteria metabolite phenyllactic acid inhibits both radial growth and sporulation of filamentous fungi. BMC Res Notes 6:464. https://doi.org/10.1186/1756-0500-6-464
Tang CM, Cohen J, Krausz T, Van Noorden S, Holden DW (1993) The alkaline protease of Aspergillus fumigatus is not a virulence determinant in two murine models of invasive pulmonary aspergillosis. Infect Immun 61(5):1650–1656. https://doi.org/10.1128/IAI.61.5.1650-1656.1993
Upadhyay SK, Mahajan L, Ramjee S, Singh Y, Basir SF, Madan T (2009) Identification and characterization of a laminin-binding protein of Aspergillus fumigatus: extracellular thaumatin domain protein (AfCalAp). J Med Microbiol 58(Pt 6):714–722. https://doi.org/10.1099/jmm.0.005991-0
Valsecchi I, Dupres V, Michel JP, Duchateau M, Matondo M, Chamilos G, Saveanu C, Guijarro JI, Aimanianda V, Lafont F, Latgé JP, Beauvais A (2019) The puzzling construction of the conidial outer layer of Aspergillus fumigatus. Cell Microbiol 21(5):e12994. https://doi.org/10.1111/cmi.12994
Vylkova S (2017) Environmental pH modulation by pathogenic fungi as a strategy to conquer the host. PLoS Pathog 13(2):e1006149. https://doi.org/10.1371/journal.ppat.1006149
Wartenberg D, Lapp K, Jacobsen ID, Dahse HM, Kniemeyer O, Heinekamp T, Brakhage AA (2011) Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein. Int J Med Microbiol 301(7):602–611. https://doi.org/10.1016/j.ijmm.2011.04.016
Wong SSW, Venugopalan LP, Beaussart A, Karnam A, Mohammed MRS, Jayapal JM, Bretagne S, Bayry J, Prajna L, Kuppamuthu D, Latgé JP, Aimanianda V (2021) Species-specific immunological reactivities depend on the cell-wall organization of the two Aspergillus, Aspergillus fumigatus and A. flavus. Front Cell Infect Microbiol 11:643312. https://doi.org/10.3389/fcimb.2021.643312
Wösten HA (2001) Hydrophobins: multipurpose proteins. Annu Rev Microbiol 55:625–646. https://doi.org/10.1146/annurev.micro.55.1.625
Yamashita N, Motoyoshi T, Nishimura A (2000) Molecular cloning of the isoamyl alcohol oxidase-encoding gene (mreA) from Aspergillus oryzae. J Biosci Bioeng 89(6):522–527. https://doi.org/10.1016/s1389-1723(00)80050-x
Yike I (2011) Fungal proteases and their pathophysiological effects. Mycopathologia 171(5):299–323. https://doi.org/10.1007/s11046-010-9386-2
Funding
This work was supported by grants from the Indo-French Center for the Promotion of Advanced Research (IFCPAR)—grant no. IFC/5403-A/2015/1012. Project assistantship to LPV was provided by IFCPAR.
Author information
Authors and Affiliations
Contributions
JMJ, DK, VA, VPN, and LP conceived and designed the research. VPN, LP, and VA contributed to the fungal isolates. LPV and JMJ performed the experiments and analyzed the data. JMJ, DK, and VA wrote the manuscript. All authors read and approved the manuscript
Corresponding author
Ethics declarations
Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Competing interests
The authors declare no competing interests.
Disclaimer
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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 7933 kb)
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Venugopalan, L.P., Aimanianda, V., Namperumalsamy, V.P. et al. Comparative proteome analysis identifies species-specific signature proteins in Aspergillus pathogens. Appl Microbiol Biotechnol 107, 4025–4040 (2023). https://doi.org/10.1007/s00253-023-12559-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-023-12559-4