Abstract
Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. One of the important questions concerning A. fumigatus is the identification of pathogenicity determinants. To obtain a comprehensive overview about the proteins produced at different physiological conditions that are related to the infectious process a proteomic approach has been applied. Here, 2-D gel electrophoresis for filamentous fungi was optimised concerning removal of interfering compounds, protein extraction and separation methods. A trichloroacetic acid-based precipitation method of proteins with their subsequent solubilisation by the use of a combination of CHAPS with a second sulfobetaine detergent gave the best results. The optimised protocol was evaluated by the analysis of the proteomes of A. fumigatus grown on two different carbon sources, i.e., glucose and ethanol. Carbon catabolite repression has not been studied in detail at the protein level in A. fumigatus yet. In addition, growth on ethanol leads to activation of the glyoxylate cycle which was shown to be essential for pathogenesis in bacteria and fungi. In A. fumigatus, differential patterns of enzymes of the gluconeogenesis, glyoxylate cycle and ethanol degradation pathway during growth on glucose and ethanol were observed.
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Bakker BM, Overkamp KM, van Maris AJA, Kötter P, Luttik MAH, van Dijken JP, Pronk JT (2001) Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol Rev 25:15–37
Beuther H-O (1988) Ethanol. In: Bergmeyer HU (eds) Methods of enzymatic analysis, vol VI. VCH, Cambridge, pp 598–606
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 7:248–254
Brakhage AA (2005) Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Curr Drug Targets 6:875–886
Brakhage AA, Langfelder K (2002) Menacing mold: the molecular biology of Aspergillus fumigatus. Annu Rev Microbiol 56:433–455
Brakhage AA, Liebmann B (2005) Aspergillus fumigatus conidial pigment and cAMP signal transduction: significance for virulence. Med Mycol 43:S75-S82
Braude AI (1986) The aspergilli. In: Braude AI, Davis CE, Fierer J (eds) Infectious diseases and medical microbiology. W.B. Saunders, Philadelphia, pp 592–597
Brock M, Buckel W (2004) On the mechanism of action of the antifungal agent propionate. Eur J Biochem 271:3227–3241
Bruneau J-M, Magnin T, Tagat E, Legrand R, Bernard M, Diaquin M, Fudali C, Latgé J-P (2001) Proteome analysis of Aspergillus fumigatus identifies glycosylphosphotidylinositol-anchored proteins associated to the cell wall biosynthesis. Electrophoresis 22:2812–2823
Carpentier SC, Witters E, Laukens K, Deckers P, Swennen R, Panis B (2005) Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5:2497–2507
Claus R, Käppeli O, Fiechter A (1982) Influence of different physiological states on carnitine-acetyl-transferase activity in Saccharomyces cerevisiae. Experientia 38:1377
Claros MG, Vincens P (1996) Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem 241:779–786
Damerval C, Devienne D, Zivy M, Thiellement H (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis 7:52–54
De Lucas JR, Valenciano S, Laborda F, Turner G (1994) Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans. Arch Microbiol 162:409–413
Ellis M (1999) Therapy of Aspergillus fumigatus-related diseases. In: Brakhage AA, Jahn B, Schmidt A (eds) Aspergillus fumigatus: biology, clinical aspects and molecular approaches to pathogenicity. Contributions to microbiology, vol 2. Karger, Basel, pp 105–129
Felenbok B, Flipphi M, Nikolaev I (2001) Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation. Prog Nucleic Acid Res Mol Biol 69:149–204
Flipphi M, Felenbok B (2004) The onset of carbon catabolic repression and interplay between specific induction and carbon catabolite repression in Aspergillus nidulans. In: Brambl R, Marzluf GA (eds) The mycota III—biochemistry and molecular biology, Springer, Berlin Heidelberg New York, pp 403–420
Gwynne DI, Buxton FP, Sibley S, Davies RW, Lockington RA, Scazzocchio C, Sealy-Lewis HM (1987) Comparison of the cis-acting control regions of two coordinately controlled genes involved in ethanol utilization in Aspergillus nidulans. Gene 51:205–216
Hoffmann B, LaPaglia SK, Kübler R, Andermann M, Eckert SE, Braus GH (2000) Developmental and metabolic regulation of the phosphoglucomutase-encoding gene, pgmB, of Aspergillus nidulans. Mol Gen Genet 262:1001–1011
Hondmann DHA, Visser J (1994) Carbon metabolism. Prog Ind Microbiol 29:61–139
Kelly JM (2004) The regulation of carbon metabolism in filamentous fungi. In: Brambl R, Marzluf GA (eds) The mycota III—biochemistry and molecular biology. Springer, Berlin Heidelberg New York, pp 386–401
Kelly JM, Hynes MJ (1981) The regulation of phosphoenolpyruvate carboxykinase and the NADP-linked malic enzyme in Aspergillus nidulans. J Gen Microbiol 123:371–375
Latgé J-P (1999) Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12:310–350
Latgé J-P, Mouyna F, Tekaia F, Beauvais A, Debeaupuis JP, Nierman W (2005) Specific molecular features in the organization and biosynthesis of the cell wall of Aspergillus fumigatus. Med Mycol 43:S15–S22
Lorenz MC, Fink GR (2001) The glyoxylate cycle is required for fungal virulence. Nature 412:83–86
Luche S, Santoni V, Rabilloud T (2003) Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 3:249–253
Mc Kinney JD, Honer zu Bentrup K, Munoz-Elias EJ, Miczak A, Chen B, Chan WT, Swenson D, Sacchettini JC, Jacobs Jr WR, Russell DG (2000) Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406:735–738
Molloy MP, Herbert BR, Williams KL, Gooley AA (1999) Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis. Electrophoresis 20:701–704
Nandakumar MP, Marten MR (2002) Comparison of lysis methods and preparation protocols for one- and two-dimensional electrophoresis of Aspergillus oryzae intracellular proteins. Electrophoresis 23:2216–2222
Nandakumar MP, Shen J, Raman B, Marten MR (2003) Solubilization of TCA precipitated microbial proteins via NaOH for two-dimensional gel electrophoresis. J Proteome Res 2:89–93
Neuhoff V, Arold N, Taube D, Ehrhardt W (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9:255–262
Niermann WC, May G, Kim HS, Anderson MJ, Chen D, Denning DW (2005) What the Aspergillus genomes have told us. Med Mycol 43:S3–S5
O’Farrel PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Olsson I, Larsson K, Palmgren R, Bjellqvist B (2002) Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension. Proteomics 2:1630–1632
Pain A, Woodward J, Quail MA et al (2004) Insight into the genome of Aspergillus fumigatus: analysis of a 922 kb region encompassing the nitrate assimilation gene cluster. Fung Genet Biol 41:443–453
Prigneau O, Porta A, Poudrier JA, Colonna-Romano S, Noel T, Maresca B (2003) Genes involved in β-oxidation, energy metabolism and glyoxylate cycle are induced by Candida albicans during macrophage infection. Yeast 20:723–730
Prigneau O, Porta A, Maresca B (2004) Candida albicans CTN gene family is induced during macrophage infection: homology, disruption and phenotypic analysis of CTN3 gene. Fung Genet Biol 41:783–793
Rabilloud T (1996) Solubilization of proteins for electrophoretic analyses. Electrophoresis 17:813–829
Rabilloud T (1998) Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis 19:758–760
Rabilloud T, Adessi C, Giraudel A, Lunardi J (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18:307–316
Schuurink R, Busink R, Hondmann DHA, Witteveen CFB, Visser J (1990) Purification and properties of NADP+-dependent glycerol dehydrogenases from Aspergillus nidulans and A. niger. J Gen Microbiol 136:1043–1050
Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal Chem 68:850–858
Shimizu M, Wariishi H (2005) Development of a sample preparation method for fungal proteomics. FEMS Microbiol Lett 247:17–22
Sims AH, Robson GD, Hoyle DC, Oliver SG, Turner G, Prade RA, Russell HH, Dunn-Coleman NS, Gent ME (2004) Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans. Fung Genet Biol 41:199–212
Steffan JS, Mc-Alister-Henn L (1992) Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase. J Biol Chem 267:24708–24715
Stemple CJ, Davis MA, Hynes MJ (1998) The facC gene of Aspergillus nidulans encodes an acetate-inducible carnitine acetyltransferase. J Bacteriol 180:6242–6251
Weidner G, d’Enfert C, Koch A, Mol PC, Brakhage AA (1998) Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′-monophosphate decarboxylase. Curr Genet 33:378–385
Wildgruber R, Reil G, Drews O, Parlar H, Görg A (2002) Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients from pH 6 to pH 12 and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteomics 2:727–732
Acknowledgements
We are grateful to Chi Wong and Geoffrey Turner of MBB at Sheffield University for introducing us in proteome analysis. Silke Steinbach is acknowledged for excellent technical assistance and Matthias Brock for critically reading of the manuscript. This research was supported by the Priority Program 1160 of the Deutsche Forschungsgemeinschaft.
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Kniemeyer, O., Lessing, F., Scheibner, O. et al. Optimisation of a 2-D gel electrophoresis protocol for the human-pathogenic fungus Aspergillus fumigatus . Curr Genet 49, 178–189 (2006). https://doi.org/10.1007/s00294-005-0047-9
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DOI: https://doi.org/10.1007/s00294-005-0047-9