Background Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species. Methodology/Principal Findings Transcriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack. Conclusion This work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.
References
[1]
Munoz P, Guinea J, Bouza E (2006) Update on invasive aspergillosis: clinical and diagnostic aspects. Clin Microbiol Infect 12: 24–39.
Bonnett CR, Cornish EJ, Harmsen AG, Burritt JB (2006) Early neutrophil recruitment and aggregation in the murine lung inhibit germination of Aspergillus fumigatus conidia. Infect Immun 74: 6528–6539.
[4]
Zarember KA, Sugui JA, Chang YC, Kwon-Chung KJ, Gallin JI (2007) Human Polymorphonuclear Leukocytes Inhibit Aspergillus fumigatus Conidial Growth by Lactoferrin-Mediated Iron Depletion. J Immunol 178: 6367–6373.
[5]
Almyroudis NG, Holland SM, Segal BH (2005) Invasive aspergillosis in primary immunodeficiencies. Med Mycol 43: S247–259.
[6]
Segal BH, Leto TL, Gallin JI, Malech HL, Holland SM (2000) Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine (Baltimore) 79: 170–200.
[7]
Segal AW (2005) How neutrophils kill microbes. Annu Rev Immunol 23: 197–223.
[8]
Rex JH, Bennett JE, Gallin JI, Malech HL, Melnick DA (1990) Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae. J Infect Dis 162: 523–528.
[9]
Langfelder K, Jahn B, Gehringer H, Schmidt A, Wanner G, et al. (1998) Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence. Med Microbiol Immunol 187: 79–89.
[10]
Tsai HF, Chang YC, Washburn RG, Wheeler MH, Kwon-Chung KJ (1998) The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence. J Bacteriol 180: 3031–3038.
[11]
Spikes S, Xu R, Nguyen CK, Chamilos G, Kontoyiannis DP, et al. (2008) Gliotoxin production in Aspergillus fumigatus contributes to host-specific differences in virulence. J Infect Dis 197: 479–486.
[12]
Sugui JA, Pardo J, Chang YC, Zarember KA, Nardone G, et al. (2007) Gliotoxin is a virulence factor of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone. Eukaryot Cell 6: 1562–1569.
[13]
Bok JW, Balajee SA, Marr KA, Andes D, Nielsen KF, et al. (2005) LaeA, a regulator of morphogenetic fungal virulence factors. Eukaryot Cell 4: 1574–1582.
[14]
Arechabala B, Coiffard C, Rivalland P, Coiffard LJ, de Roeck-Holtzhauer Y (1999) Comparison of cytotoxicity of various surfactants tested on normal human fibroblast cultures using the neutral red test, MTT assay and LDH release. J Appl Toxicol 19: 163–165.
[15]
Babich H, Borenfreund E (1991) Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay. Appl Environ Microbiol 57: 2101–2103.
[16]
Mason DL, Wilson CL (1979) Cytochemical and biochemical identification of lysosomes in Cryptococcus neoformans. Mycopathologia 68: 183–190.
[17]
Schadeck RJ, Randi MA, de Freitas Buchi D, Leite B (2003) Vacuolar system of ungerminated Colletotrichum graminicola conidia: convergence of autophagic and endocytic pathways. FEMS Microbiol Lett 218: 277–283.
[18]
Weber RW, Wakley GE, Pitt D (1999) Histochemical and ultrastructural characterization of vacuoles and spherosomes as components of the lytic system in hyphae of the fungus Botrytis cinerea. Histochem J 31: 293–301.
[19]
Urban CF, Reichard U, Brinkmann V, Zychlinsky A (2006) Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol 8: 668–676.
[20]
Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, et al. (2005) Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438: 1151–1156.
[21]
Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116–5121.
[22]
Sugui JA, Chang YC, Kwon-Chung KJ (2005) Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Appl Environ Microbiol 71: 1798–1802.
[23]
Hynes MJ, Murray SL, Duncan A, Khew GS, Davis MA (2006) Regulatory genes controlling fatty acid catabolism and peroxisomal functions in the filamentous fungus Aspergillus nidulans. Eukaryot Cell 5: 794–805.
[24]
Gurvitz A, Rottensteiner H (2006) The biochemistry of oleate induction: transcriptional upregulation and peroxisome proliferation. Biochim Biophys Acta 1763: 1392–1402.
Hearn VM, Wilson EV, Mackenzie DW (1992) Analysis of Aspergillus fumigatus catalases possessing antigenic activity. J Med Microbiol 36: 61–67.
[27]
Paris S, Wysong D, Debeaupuis JP, Shibuya K, Philippe B, et al. (2003) Catalases of Aspergillus fumigatus. Infect Immun 71: 3551–3562.
[28]
Yamamoto A, Ueda J, Yamamoto N, Hashikawa N, Sakurai H (2007) Role of heat shock transcription factor in Saccharomyces cerevisiae oxidative stress response. Eukaryot Cell 6: 1373–1379.
[29]
Magnani T, Soriani FM, Martins VP, Nascimento AM, Tudella VG, et al. (2007) Cloning and functional expression of the mitochondrial alternative oxidase of Aspergillus fumigatus and its induction by oxidative stress. FEMS Microbiol Lett 271: 230–238.
[30]
Lorenz MC, Bender JA, Fink GR (2004) Transcriptional response of Candida albicans upon internalization by macrophages. Eukaryot Cell 3: 1076–1087.
[31]
Tavares AH, Silva SS, Dantas A, Campos EG, Andrade RV, et al. (2007) Early transcriptional response of Paracoccidioides brasiliensis upon internalization by murine macrophages. Microbes Infect 9: 583–590.
[32]
Derengowski LS, Tavares AH, Silva S, Procopio LS, Felipe MS, et al. (2008) Upregulation of glyoxylate cycle genes upon Paracoccidioides brasiliensis internalization by murine macrophages and in vitro nutritional stress condition. Med Mycol 46: 125–134.
[33]
Fradin C, De Groot P, MacCallum D, Schaller M, Klis F, et al. (2005) Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood. Mol Microbiol 56: 397–415.
Bojunga N, Kotter P, Entian KD (1998) The succinate/fumarate transporter Acr1p of Saccharomyces cerevisiae is part of the gluconeogenic pathway and its expression is regulated by Cat8p. Mol Gen Genet 260: 453–461.
[36]
Palmieri L, Lasorsa FM, De Palma A, Palmieri F, Runswick MJ, et al. (1997) Identification of the yeast ACR1 gene product as a succinate-fumarate transporter essential for growth on ethanol or acetate. FEBS Lett 417: 114–118.
[37]
Prigneau O, Porta A, Poudrier JA, Colonna-Romano S, Noel T, et al. (2003) Genes involved in beta-oxidation, energy metabolism and glyoxylate cycle are induced by Candida albicans during macrophage infection. Yeast 20: 723–730.
[38]
Ebel F, Schwienbacher M, Beyer J, Heesemann J, Brakhage AA, et al. (2006) Analysis of the regulation, expression, and localisation of the isocitrate lyase from Aspergillus fumigatus, a potential target for antifungal drug development. Fungal Genet Biol 43: 476–489.
[39]
Schobel F, Ibrahim-Granet O, Ave P, Latge JP, Brakhage AA, et al. (2006) Aspergillus fumigatus does not require fatty acid metabolism via isocitrate lyase for development of invasive aspergillosis. Infect Immun 74: 1237–1244.
[40]
Lorenz MC, Fink GR (2001) The glyoxylate cycle is required for fungal virulence. Nature 412: 83–86.
[41]
Dinauer MC, Lekstrom-Himes JA, Dale DC (2000) Inherited Neutrophil Disorders: Molecular Basis and New Therapies. pp. 303–318.
[42]
Calera JA, Paris S, Monod M, Hamilton AJ, Debeaupuis JP, et al. (1997) Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus. Infect Immun 65: 4718–4724.
[43]
Chang YC, Segal BH, Holland SM, Miller GF, Kwon-Chung KJ (1998) Virulence of catalase-deficient aspergillus nidulans in p47(phox)-/- mice. Implications for fungal pathogenicity and host defense in chronic granulomatous disease. J Clin Invest 101: 1843–1850.
[44]
Yun CW, Bauler M, Moore RE, Klebba PE, Philpott CC (2001) The role of the FRE family of plasma membrane reductases in the uptake of siderophore-iron in Saccharomyces cerevisiae. J Biol Chem 276: 10218–10223.
[45]
Dancis A, Klausner RD, Hinnebusch AG, Barriocanal JG (1990) Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae. Mol Cell Biol 10: 2294–2301.
[46]
Anderson GJ, Lesuisse E, Dancis A, Roman DG, Labbe P, et al. (1992) Ferric iron reduction and iron assimilation in Saccharomyces cerevisiae. J Inorg Biochem 47: 249–255.
[47]
Georgatsou E, Alexandraki D (1994) Two distinctly regulated genes are required for ferric reduction, the first step of iron uptake in Saccharomyces cerevisiae. Mol Cell Biol 14: 3065–3073.
[48]
Askwith C, Eide D, Van Ho A, Bernard PS, Li L, et al. (1994) The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake. Cell 76: 403–410.
[49]
Dancis A, Yuan DS, Haile D, Askwith C, Eide D, et al. (1994) Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport. Cell 76: 393–402.
[50]
Schrettl M, Bignell E, Kragl C, Joechl C, Rogers T, et al. (2004) Siderophore biosynthesis but not reductive iron assimilation is essential for Aspergillus fumigatus virulence. J Exp Med 200: 1213–1219.
[51]
Wasylnka JA, Hissen AH, Wan AN, Moore MM (2005) Intracellular and extracellular growth of Aspergillus fumigatus. Med Mycol 43: Suppl 1S27–30.
[52]
Hissen AH, Wan AN, Warwas ML, Pinto LJ, Moore MM (2005) The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence. Infect Immun 73: 5493–5503.
[53]
Blaiseau PL, Lesuisse E, Camadro JM (2001) Aft2p, a novel iron-regulated transcription activator that modulates, with Aft1p, intracellular iron use and resistance to oxidative stress in yeast. J Biol Chem 276: 34221–34226.
