AiM  Vol.8 No.1 , January 2018
Proteomic Differences between Azole-Susceptible and -Resistant Aspergillus fumigatus Strains
Abstract: Background: Azole-resistance is increasingly reported in Aspergillus fumigatus infections. It remains challenging to rapidly assess antifungal susceptibility to initiate the appropriate therapy. The aim of this study was to map the proteomic differences of azole-susceptible and -resistant strains. Methods: Proteomic studies were performed with ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS). Results: UPLC-MS/MS detected 7899 peptides, of which 1792 peptides had a significantly different abundance (p < 0.05) between resistant and susceptible strains. The discriminating proteins were identified and provide an interesting tool for future research into A. fumigatus resistance. Conclusions: UPLC-MS/MS provided proof-of-concept that the proteome of azole-resistant A. fumigatus is diverse enough to serve as a diagnostic tool.
Cite this paper: Vermeulen, E. , Carpentier, S. , Kniemeyer, O. , Sillen, M. , Maertens, J. and Lagrou, K. (2018) Proteomic Differences between Azole-Susceptible and -Resistant Aspergillus fumigatus Strains. Advances in Microbiology, 8, 77-99. doi: 10.4236/aim.2018.81007.

[1]   Fedorova, N.D., Khaldi, N., Joardar, V.S., et al. (2008) Genomic Islands in the Pathogenic Filamentous Fungus Aspergillus fumigatus. PLoS Genetics, 4, e1000046.

[2]   Van Pamel, E., Daeseleire, E., De Clercq, N., Herman, L., Verbeken, A., Heyndrickx, M. and Vlaemynck, G. (2012) Restriction Analysis of an Amplified rodA Gene Fragment to Distinguish Aspergillus fumigatus var.ellipticus from Aspergillus fumigatus var.fumigatus. FEMS Microbiology Letters, 333, 153-159.

[3]   Marti, N., Fonteyne, P.A. and Nolard, N. (2002) Multilocus Sequence Analysis of Aspergillus fumigatus Diversity. Submitted (MAR) to the EMBL/GenBank/DDBJ Databases.

[4]   Aimanianda, V., Bayry, J., Bozza, S., et al. (2009), Surface, Hydrophobin, Prevents, Immune Recognition of Airborne Fungal Spores. Nature, 460, 1117-1121.

[5]   Carrion Sde, J., Leal Jr., S.M., Ghannom, M.A., Aimanianda, V., Latgé, J.P. and Pearlman, E. (2013) The Roda Hydrophobin on Aspergillus fumigatus Spores Masks Dectin-1- and Dectin-2-Dependent Responses and Enhances Fungal Survival In Vivo. The Journal of Immunology, 191, 2581-2588.

[6]   Campoli, P., Perlin, D.S., Kristof, A.S., White, T.C., Filler, S.G. and Sheppard, D.C. (2013) Pharmacokinetics of Posaconazole within Epithelial Cells and Fungi: Insights into Potential Mechanisms of Action during Treatment and Prophylaxis. The Journal of Infectious Diseases, 208, 1717-1728.

[7]   Bruns, S., Kniemeyer, O., Hasenberg, M., et al. (2010) Production of Extracellular Traps against Aspergillus fumigatus In Vitro and in Infected Lung Tissue Is Dependent on Invading Neutrophils and Influenced by Hydrophobin RodA. PLoS Pathogens, 6, e1000873.

[8]   Nierman, W.C., Pain, A., Anderson, M.J., et al. (2005) Genomic Sequence of the Pathogenic and Allergenic Filamentous Fungus Aspergillus fumigatus. Nature, 438, 1151-1156.

[9]   Wood, V., Gwilliam, R., Rajandream, M.A., et al. (2002) The Genome Sequence of Schizosaccharomyces pombe. Nature, 415, 871-880.

[10]   Silar, P., Koll, F. and Rossignol, M. (1997) Cytosolic Ribosomal Mutations That Abolish Accumulation of Circular Intron in the Mitochondria without Preventing Senescence of Podospora anserina. Genetics, 145, 697-705.

[11]   Galagan, J.E., Calvo, S.E., Cuomo, C., et al. (2005) Sequencing of Aspergillus nidulans and Comparative Analysis with A. fumigatus and A. oryzae. Nature, 438, 1105-1115.

[12]   Cerqueira, G.C., Arnaud, M.B., Inglis, D.O., et al. (2013) The Aspergillus Genome Database: Multispecies Curation and Incorporation of RNA-Seq Data to Improve Structural Gene Annotations. Nucleic Acids Research, 42, D705-D710.

[13]   Links, M.G., Dumonceaux, T.J., Hemmingsen, S.M. and Hill, J.E. (2012) The Chaperonin-60 Universal Target Is a Barcode for Bacteria That Enables De Novo Assembly of Metagenomic Sequence Data. PLoS One, 7, e49755.

[14]   Raggam, R.B., Salzer, H.J., Marth, E., Heiling, B., Paulitsch, A.H. and Buzina, W. (2011) Molecular Detection and Characterisation of Fungal Heat Shock Protein 60. Mycoses, 54, e394-e399.

[15]   Mittelman, D., Sykoudis, K., Hersh, M., Lin, Y. and Wilson, J.H. (2010) Hsp90 Modulates CAG Repeat Instability in Human Cells. Cell Stress and Chaperones, 15, 753-759.

[16]   Gemayel, R., Vinces, M.D., Legendre, M. and Verstrepen, K.J. (2010) Variable Tandem Repeats Accelerate Evolution of Coding and Regulatory Sequences. Annual Review of Genetics, 44, 445-477.

[17]   Hand, R.A., Jia, N., Bard, M. and Craven, R.J. (2003) Saccharomyces cerevisiae Dap1p, a Novel DNA Damage Response Protein Related to the Mammalian Mem-brane-Associated Progesterone Receptor. Eukaryotic Cell, 2, 306-317.

[18]   Hagiwara, D., Takahashi, H., Watanabe, A., Takahashi-Nakaguchi, A., Kawamoto, S., Kamei, K. and Gonoi, T. (2014) Whole-Genome Comparison of Aspergillus fumigatus Strains Serially Isolated from Patients with Aspergillosis. Journal of Clinical Microbiology, 52, 4202-4209.

[19]   Holdom, M.D., Hay, R.J. and Hamilton, A.J. (1996) The Cu,Zn Superoxide Dismutases of Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus: Purification and Biochemical Comparison with the Aspergillus fumigatus Cu,Zn Superoxide Dismutase. Infection and Immunity, 64, 3326-3332.

[20]   Leal Jr., S.M., Vareechon, C., Cowden, S., Cobb, B.A., Latgé, J.P., Momany, M. and Pearlman, E. (2012) Fungal Antioxidant Pathways Promote Survival against Neutrophils during Infection. The Journal of Clinical Investigation, 122, 2482-2498.

[21]   Lambou, K., Lamarre, C., Beau, R., Dufour, N. and Latge, J.P. (2010) Functional Analysis of the Superoxide Dismutase Family in Aspergillus fumigatus. Molecular Microbiology, 75, 910-923.

[22]   Abadio, A.K., Kioshima, E.S., Teixeira, M.M., Martins, N.F., Maigret, B. and Felipe, M.S. (2011) Comparative Genomics Allowed the Identification of Drug Targets against Human Fungal Pathogens. BMC Genomics, 12, 75.

[23]   Shi, L.N., Li, F.Q., Lu, J.F., et al. (2012) Antibody Specific to Thioredoxin Reductase as a New Biomarker for Serodiagnosis of Invasive Aspergillosis in Non-Neutropenic Patients. Clinica Chimica Acta, 413, 938-943.

[24]   Glaser, A.G., Menz, G., Kirsch, A.I., Zeller, S., Crameri, R. and Rhyner, C. (2008) Auto- and Cross-Reactivity to Thioredoxin Allergens in Allergic Bronchopulmonary Aspergillosis. Allergy, 63, 1617-1623.

[25]   Alarco, A.M. and Raymond, M. (1999) The bZip Transcription Factor Cap1p Is Involved in Multidrug Resistance and Oxidative Stress Response in Candida albicans. Journal of Bacteriology, 181, 700-708.

[26]   Schwienbacher, M., Weig, M., Thies, S., Regula, J.T., Heesemann, J. and Ebel, F. (2005) Analysis of The Major Proteins Secreted by the Human Opportunistic Pathogen Aspergillus fumigatus under In Vitro Conditions. Medical Mycology, 43, 623-630.

[27]   Sharpton, T.J., Stajich, J.E., Rounsley, S.D., et al. (2009) Comparative Genomic Analyses of the Human Fungal Pathogens Coccidioides and Their Relatives. Genome Research, 19, 1722-1731.

[28]   Liu, D., Zhang, R., Yang, X., Zhang, Z., Song, S., Miao, Y. and Shen, Q. (2012) Characterization of a Thermostable β-Glucosidase from Aspergillus fumigatus Z5, and Its Functional Expression in Pichia pastoris X33. Microbial Cell Factories, 11, 25.

[29]   Potenza, L., Vallerini, D., Barozzi, P., et al. (2013) Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients. PLoS One, 8, e74326.

[30]   Zhao, Y., Paderu, P., Park, S., Dukhan, A., Senter, M. and Perlin, D.S. (2012) Expression Turnover Profiling to Monitor the Antifungal Activities of Amphotericin B, Voriconazole, and Micafungin against Aspergillus fumigatus. Antimicrobial Agents and Chemotherapy, 56, 2770-2772.

[31]   Thompson, L.M., Sutherland, P., Steffan, J.S. and McAlister-Henn, L. (1988) Gene Sequence and Primary Structure of Mitochondrial Malate Dehydrogenase from Saccharomyces cerevisiae. Biochemistry, 27, 8393-8400.

[32]   Zhang, X., Wang, Y., Chi, W., Shi, Y., Chen, S., Lin, D. and Jin, Y. (2014) Metalloprotease Genes of Trichophyton Mentagrophytes Are Important for Pathogenicity. Medical Mycology, 52, 36-45.

[33]   Rappleye, C.A. and Goldman, W.E. (2006) Defining Virulence Genes in the Dimorphic Fungi. Annual Review of Microbiology, 60, 281-303.

[34]   Lessing, F., Kniemeyer, O., Wozniok, I., Loeffler, J., Kurzai, O., Haertl, A. and Brakhage, A.A. (2007) The Aspergillus fumigatus Transcriptional Regulator AfYap1 Represents the Major Regulator for Defense against Reactive Oxygen Intermediates but Is Dispensable for Pathogenicity in an Intranasal Mouse Infection Model. Eukaryotic Cell, 6, 2290-2302.

[35]   Asif, A.R., Oellerich, M., Amstrong, V.W., Gross, U. and Reichard, U. (2010) Analysis of the Cellular Aspergillus fumigatus Proteome That Reacts with Sera from Rabbits Developing an Acquired Immunity after Experimental Aspergillosis. Electrophoresis, 31, 1947-1958.

[36]   Kumar, A., Ahmed, R., Singh, P.K. and Shukla, P.K. (2011) Identification of Virulence Factors and Diagnostic Markers Using Immunosecretome of Aspergillus fumigatus. Journal of Proteomics, 74, 1104-1112.

[37]   Oosthuizen, J.L., Gomez, P., Ruan, J., Hackett, T.L., Moore, M.M., Knight, D.A. and Tebbutt, S.J. (2011) Dual Organism Transcriptomics of Airway Epithelial Cells Interacting with Conidia of Aspergillus fumigatus. PLoS One, 6, e20527.

[38]   Da Silva Ferreira, M.E., Malavazi, I., Savoldi, M., et al. (2006) Transcriptome Analysis of Aspergillus fumigatus Exposed to Voriconazole. Current Genetics, 50, 32-44.

[39]   Xu, D., Jiang, B., Ketela, T., et al. (2007) Genome-Wide Fitness Test and Mechanism-of-Action Studies of Inhibitory Compounds in Candida albicans. PLoS Pathogens, 3, e92.

[40]   Tsitsigiannis, D.I., Bok, J.W., Andes, D., Nielsen, K.F., Frisvad, J.C. and Keller, N.P. (2005) Aspergillus Cyclooxygenase-Like Enzymes Are Associated with Prostaglandin Production and Virulence. Infection and Immunity, 73, 4548-4559.

[41]   Li, Y.J. and Jin, C. (2004) Mannosidase I from Aspergillus fumigatus YJ-407. Submitted to the EMBL/GenBank/DDBJ Databases.

[42]   Sugui, J.A., Kim, H.S., Zarember, K.A., Chang, Y.C., Gallin, J.I., Nierman, W.C. and Kwon-Chung, K.J. (2008) Genes Differentially Expressed in Conidia and Hyphae of Aspergillus fumigatus upon Exposure to Human Neutrophils. PLoS One, 3, e2655.

[43]   Willger, S.D., Puttikamonkul, S., Kim, K.H., et al. (2008) A Sterol-Regulatory Element Binding Protein Is Required for Cell Polarity, Hypoxia Adaptation, Azole Drug Resistance, and Virulence in Aspergillus fumigatus. PLoS Pathogens, 4, e1000200.

[44]   Kuboi, S., Ishimaru, T., Tamada, S., Bernard, E.M., Perlin, D.S. and Armstrong, D. (2013) Molecular Characterization of AfuFleA, An L-Fucose-Specific Lectin from Aspergillus fumigatus. Journal of Infection and Chemotherapy, 19, 1021-1028.

[45]   Ishimaru, T., Kuboi, S., Bernard, E.M., Tamada, S., Tong, W., Soteropuolos, P., Perlin, D.S. and Armstrong, D. (2002) Aspergillus fumigatus Fucose-Specific Lectin (AFL1) Gene, Complete Cds. Submitted to the EMBL/GenBank/DDBJ databases.

[46]   Houser, J., Komarek, J., Kostlanova, N., et al. (2013) A Soluble Fucose-Specific Lectin from Aspergillus fumigatus Conidia—Structure, Specificity and Possible Role in Fungal Pathogenicity. PLoS One, 8, e83077.

[47]   Kotiadis, V.N., Leadsham, J.E., Bastow, E.L., et al. (2012) Identification of New Surfaces of Cofilin That Link Mitochondrial Function to the Control of Multi-Drug Resistance. Journal of Cell Science, 125, 2288-2299.

[48]   Blatzer, M., Barker, B.M., Willger, S.D., et al. (2011) SREBP Coordinates Iron and Ergosterol Homeostasis to Mediate Triazole Drug and Hypoxia Responses in the Human Fungal Pathogen Aspergillus fumigatus. PLoS Genetics, 7, e1002374.

[49]   Dujon, B., Sherman, D., Fischer, G., et al. (2004) Genome Evolution in Yeasts. Nature, 430, 35-44.

[50]   Tkach, J.M., Yimit, A., Lee, A.Y., et al. (2012) Dissecting DNA Damage Response Pathways by Analysing Protein Localization and Abundance Changes during DNA Replication Stress. Nature Cell Biology, 14, 966-976.

[51]   Rambach, G., Dum, D., Mohsenipour, I., Hagleitner, M., Würzner, R., Lass-Flörl, C. and Speth, C. (2010) Secretion of a Fungal Protease Represents a Complement Evasion Mechanism in Cerebral Aspergillosis. Molecular Immunology, 47, 1438-1449.

[52]   Behnsen, J., Lessing, F., Schindler, S., et al. (2010) Secreted Aspergillus fumigatus Protease Alp1 Degrades Human Complement Proteins C3, C4, and C5. Infection and Immunity, 78, 3585-3594.