Source: http://www.asmscience.org/content/book/10.1128/9781555815523.ch05
Timestamp: 2019-04-21 20:18:27+00:00

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This chapter discusses recent developments in the identification of essential genes and validation of potential antifungal drug targets in Aspergillus fumigatus. Essential genes were identified based on (i) the inability to construct haploid insertional mutants or (ii) identification of temperature-sensitive conditional mutants. The compendium of recently defined conserved essential genes in Saccharomyces cerevisiae, Candida albicans, and other fungi has provided important insights for predicting essential genes in A. fumigatus. Essential genes that are required for fungal survival and growth provide potential antifungal drug targets. A. fumigatus genes which have been experimentally demonstrated to be essential for growth are summarized. This essential gene set includes genes involved in various biological and biochemical functions, such as amino acid, cell wall, ergosterol, heme, and lipid biosynthesis, as well as cell cycle control, cellular metabolism, protein transport, ribosome biogenesis, and RNA splicing. Additional A. fumigatus essential genes involved in ergosterol biosynthesis include ERG10, ERG12, ERG7, ERG8, and ERG20 and as such, provide new targets for therapeutic intervention. Currently, identification of A. fumigatus essential genes largely depends on the following four approaches: conventional gene deletion and disruption, parasexual genetics, RNAi knockdown, and conditional promoter replacement strategies. Completion of the A. fumigatus genome sequence, however, combined with current molecular genetic strategies and their inevitable refinements, has now made large-scale genetic analysis of A. fumigatus possible for the first time, thus expanding our knowledge of its biology, pathogenesis, and potential antifungal targets.
Outline of the GRACE method of target validation in C. albicans. Step 1: A heterozygote strain of the target gene was constructed by transforming a wild-type C. albicans strain with a PCR-generated HIS3 disruption cassette flanked with homologous sequences to precisely delete one copy of the target gene. Step 2: The heterozygote strain obtained in step 1 was further transformed with a PCR-generated conditional promoter replacement cassette. Each cassette contains a SAT-1 dominant selectable marker and a conditional promoter (pTet) flanked with homologous DNA sequences to precisely replace the endogenous promoter of the remaining wild-type allele with pTet. Gene essentiality of the target gene was directly assessed by comparing the growth phenotype under inducing and repressing conditions ( Roemer et al., 2003 ).
Schematic overview of gene disruption and deletion methods. (A) Schematic representation of a gene disruption event. The open arrow represents the ORF of a gene of interest. A gene disruption plasmid was constructed by cloning a truncated fragment of the target gene into a plasmid containing a selectable marker. Following a single-crossover homologous recombination, the plasmid integrates into the target gene, leading to a disruption of the gene. (B) Schematic representation of the gene deletion method. The open arrow represents the ORF of a gene of interest. A gene deletion cassette containing a selectable marker flanked with appropriate homologous DNA sequence was used to transform A. fumigatus. Following a double-crossover homologous recombination, the ORF of the target gene was precisely replaced by the selectable marker.
Schematic overview of the parasexual strategy. A diploid (heterozygous) A. fumigatus strain was first created by gene disruption or transposon mutagenesis and contains one inactivated allele of the target gene (gene X) as well as a wild-type allele. The heterozygous strain is used to perform haploidization analysis using benomyl as an inducer on selective and nonselective medium in two independent tests. The haploidization process will result in two subpopulations of haploid cells: one bearing the inactivated allele of the target gene and one bearing a wild-type allele. If gene X is essential for A. fumigatus growth, haploid progenies cannot be obtained from selective medium, as haploids with the inactivated allele will not be viable and haploids with the wild-type allele lack the selectable marker. Replicated from Firon and d’Enfert (2002) with permission from the publisher and the authors.
Schematic overview of the RNAi method. (A) Representative example of the RNAi cassette used to silence the target gene in A. fumigatus. The RNAi cassette was constructed with inverted repeats of 500 bp of the coding region of the target gene separated by a spacer segment of GFP sequence. (B) Representative example of the AMA1 -based RNAi cassette.
Conditional promoter replacement strategy. (A) Schematic overview of the pNiiA-CPR strategy. A conditional promoter replacement cassette containing a PyrG selectable marker and a pNiiA conditional promoter flanked with 1.5 kb of homologous DNA sequence (L-arm and R-arm) was used to transform the A. fumigatus CEA17 strain (PyrG -). Following homologous recombination, the endogenous promoter of the target gene was precisely replaced by the pNiiA condition promoter ( Hu et al., 2007 ). (B) Representative example of gene essentiality validation with a pNiiA-MET2 mutant. The pNiiA-MET2 mutant displayed a no-growth phenotype under repressing conditions, suggesting its essential role for growth. Images are reprinted from Hu et al. (2007) with permission from the publisher and the authors.
In vivo validation of gene essentiality using pNiiA-CPR mutants. (A) ICR male mice were immunocompromised by administrating cyclophosphamide at 150 mg/kg of body weight twice prior to infection and then 100 mg/kg twice a week after infection. Approximately 105 viable conidia from individual pNHA-CPR mutants were injected into the tail vein of immunocompromised mice (five mice per group). CEA10 (wild-type) and CEA17 (a PyrG–auxotroph of CEA10) were included as controls for virulence and avirulence, respectively. (B) Genetic inactivation of the ERG11 gene family promotes avirulence in an immunocompromised murine model of systemic infection. Pathogenesis of ergllAA, ergllBA, and an ERG11 double mutant (ergl 1BA pNHA-ERGl 1A) was similarly analyzed but over a longer postinfection period (22 days), and animal survival was compared to CEA10 and CEA17 control strains. Figures are reprinted from Hu et al. (2007) with permission from the publisher and the authors.
1. Agrawal, N.,, P. V. Dasaradhi,, A. Mohmmed,, P. Malhotra,, R. K. Bhat-nagar, and, S. K. Mukherjee. 2003. RNA interference: biology, mechanism, and applications. Microbiol. Mol. Biol.Rev. 67: 657– 685.
2. Ahting, U.,, M. Thieffry,, H. Engelherdt,, R. Hegerl,, W. Neupert, and, S. Nussberger. 2001. Tom40,the poreforming component of the protein-conducting TOM channel in the outer memberane of mitochondria. J. Cell Biol. 153: 1151– 1160.
3. Akins, R. A., 2005. An update on antifungal targets and mechanisms of resistance in Candida albicans. Med. Mycol. 43: 285– 318.
4. Aleksenko, A. Y., and, A. J. Clutterbuck. 1995. Recombinational stability of replicating plasmids in Aspergillus nidulans during transformation, vegetative growth and sexual reproduction. Curr. Genet. 28: 87– 93.
5. Aleksenko, A.,, I. Nikolaev,, Y. Vinetski, and, A. J. Clutterbuck. 1996. Gene expression from replicating plasmids in Aspergillus nidulans. Mol. Gen. Genet. 253: 242– 246.
6. Amaar, Y. G.,and, M. M. Moore. 1998. Mapping of the nitrate-assimilation gene cluster (crnA-niiA-niaD) and characterization of the nitrite reductase gene (niiA) in the opportunistic fungal pathogen Aspergillus fumigatus. Curr. Genet. 33: 206– 215.
7. Baker, K. P.,, A. Schaniel,, D. Vestweber, and, G. Schatz. 1990. A yeast mitochondrial outer membrane protein essential for protein import and cell viability. Nature 348: 605– 609.
8. Baulcombe,, D. 2001. RNA silencing. Diced defence. Nature 409: 295– 296.
9. Beauvais, A.,, D. Maubon,, S. Park,, W. Morelle,, M. Tanguy,, M. Huerre,, D. S. Perlin, and, J. P. Latgé. 2005. Two α(1-3) glucan synthases with different functions in Aspergillus fumigatus. Appl. Environ. Microbiol. 71: 1531– 1538.
10. Bhabhra,, R.,, M. D. Miley,, E. Mylonakis,, D. Boettner,, J. Fortwendel,, J. C. Panepinto,, M. Postow,, J. C. Rhodes, and, D. S. Askew. 2004. Disruption of the Aspergillus fumigatus gene encoding nucleolar protein CgrA impairs thermotolerant growth and reduces virulence. Infect. Immun. 72: 4731– 4740.
11. Bok, J. W.,, D. Chung,, S. A. Balajee,, K. A. Marr,, D. Andes,, K. F. Nielsen,, J. C. Frisvad,, K. A. Kirby, and, N. P. Keller. 2006. GHZ, a transcriptional regulator of gliotoxin biosynthesis, contributes to Aspergillus fumigatus virulence. Infect. Immun. 74: 6761– 6768.
12. Brakhage,, A. A. 2005. Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Curr. Drug Targets 6: 875– 886.
13. Brakhage,, A. A.,and, K. Langfelder. 2002. Menacing mold: the molecular biology of Aspergillus fumigatus. Annu. Rev. Microbiol. 56: 433– 455.
14. Bromley,, M.,, C. Gordon,, N. Rovira-Graells, and, J. Oliver. 2006. The Aspergillus fumigatus cellobiohydrolase B (cbhB) promoter is tightly regulated and can be exploited for controlled protein expression and RNAi. FEMS Microbiol. Lett. 264: 246– 254.
15. Brookman,, J. L.,and, D. W. Denning 2000. Molecular genetics in Aspergillus fumigatus. Curr. Opin. Microbiol. 3: 468– 474.
16. Brown,, J. S.,, A. Aufauvre-Brown, J. Brown,, J. M. Jennings,, H. Arst, Jr., and, D. W. Holden. 2000. Signature-tagged and directed mutagenesis identify PABA synthetase as essential for Aspergillus fumigatus pathogenicity. Mol. Microbiol. 36: 1371– 1380.
17. Bruno,, V. M.,, S. Kalachikov,, R. Subaran,, C. J. Nobile,, C. Kyratsous, and, A. P. Mitchell. 2006. Control of the C. albicans cell wall damage response by transcriptional regulator Cas5. PLoS Pathog. 2: e21.
18. Cairns,, B. R.,, Y. Lorch,, Y. Li,, M. Zhang,, L. Lacomis,, H. Erdjument-Bromage, P. Tempst,, J. Du,, B. Laurent, and, R. D. Kornberg. 1996. RSC, an essential, abundant chromatin-remodeling complex. Cell 87: 1249– 1260.
19. Caplen,, N. J.,, S. Parrish,, F. Imani,, A. Fire, and, R. A. Morgan. 2001. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98: 9742– 9747.
20. Charbonneau, C, I. Fournier,, S. Dufresne,, J. Barwicz, and, P. Tan-crede. 2001. The interactions of amphotericin B with various sterols in relation to its possible use in anticancer therapy. Biophys. Chem. 91: 125– 133.
21. Chittur,, S., and, R. Griffith. 2002. Multisubstrate analogue inhibitors of glucosamine-6-phosphate synthase from Candida albicans. Bioorg. Med. Chem. Lett. 12: 2639– 2642.
22. Chmara,, H.,, S. Milewski,, R. Andruszkiewicz,, F. Mignini, and, E. Bo-rowski. 1998. Antibacterial action of dipeptides containing an inhibitor of glucosamine-6-phosphate isomerase. Microbiology 144: 1349– 1358.
23. Clutterbuck, A. J. 1992. Sexual and parasexual genetics of Aspergillus species. Biotechnology 23: 3– 18.
24. Colot,, H. V.,, G. Park,, G. E. Turner,, C. Ringelberg,, C. M. Crew,, L. Litvinkova,, R. L. Weiss,, K. A. Borkovich, and, J. C. Dunlap. 2006. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc. Natl. Acad. Sci. USA 103: 10352– 10357.
25. Costa,, S., and, M. Nucci. 2001. Can we decrease amphotericin ne-phrotoxicity? Curr. Opin. Crit. Care 7: 379– 383.
26. Cramer,, R. A., Jr., M. P. Gamcsik,, R. M. Brooking,, L. K. Najvar,, W. R. Kirkpatrick,, T. F. Patterson,, C. J. Balibar,, J. R. Graybill,, J. R. Perfect,, S. N. Abraham, and, W. J. Steinbach. 2006. Disruption of a nonribosomal peptide synthetase in Aspergillus fumigatus eliminates gliotoxin production. Eukaryot. Cell 5: 972– 980.
27. Damelin,, M.,, I. Simon,, T. I. Moy,, B. Wilson,, S. Komili,, P. Tempst,, F. P. Roth,, R. A. Young,, B. R. Cairns, and, P. A. Silver. 2002. The genome-wide localization of Rsc9, a component of the RSC chromatin-remodeling complex, changes in response to stress. Mol. Cell 9: 563– 573.
28. da Silva Ferreira, M. E.,, M. R. Kress,, M. Savoldi,, M. H. Goldman,, A. Hard,, T. Heinekamp,, A. A. Brakhage, and, G. H. Goldman. 2006. The akuB KU80 mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot. Cell 5: 207– 211.
29. Davydenko,, S. G.,, J. K. Juselius,, T. Munder,, E. Bogengruber,, J. Jantti, and, S. Keranen. 2004. Screening for novel essential genes of Sac-charomyces cerevisiae involved in protein secretion. Yeast 21: 463– 471.
30. De Backer, M. D.,, B. Nelissen,, M. Logghe,, J. Viaene,, I. Loonen,, S. Vandoninck,, R. de Hoogt,, S. Dewaele,, F. A. Simons,, P. Verhasselt,, G. Vanhoof,, R. Contreras, and, W. H. Luyten. 2001. An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans. Nat. Biotechnol. 19: 235– 241.
31. d’Enfert, C. 1996. Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5′-decarboxylase gene, pyrG, as a unique transformation marker. Curr. Genet. 30: 76– 82.
32. d’Enfert, C,, M. Diaquin,, A. Delit,, N. Wuscher,, J. P. Debeaupuis,, M. Huerre, and, J. P. Latgé. 1996. Attenuated virulence of uridine-uracil auxotrophs of Aspergillus fumigatus. Infect. Immun. 64: 4401– 4405.
33. Denning, D. W. 1998. Invasive aspergillosis. Clin. Infect. Dis. 26: 781– 803.
34. Dickson,, R. C, and, R. L. Lester. 1999. Metabolism and selected functions of sphingolipids in the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 1438: 305– 321.
35. Dujardin,, G.,, M. Kermorgant,, P. P. Slonimski, and, H. Boucherie. 1994. Cloning and sequencing of the GMP synthetase-encoding gene of Saccharomyces cerevisiae. Gene 139: 127– 132.
36. Felenbok,, B.,, M. Flipphi, and, I. Nikolaev. 2001. Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation. Prog.Nucleic Acid Res. Mol. Biol. 69: 149– 204.
37. Fire,, A. Z. 2007. Gene silencing by double-stranded RNA., Cell Death Differ. 14: 1998– 2012.
38. Firon,, A., and, C. d’Enfert. 2002. Identifying essential genes in fungal pathogens of humans. Trends Microbiol. 10: 456– 462.
39. Firon,, A.,, F. Villalba,, R. Beffa, and, C. D’Enfert. 2003. Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis. Eukaryot. Cell 2: 247– 255.
40. Fortwendel,, J. R.,, W. Zhao,, R. Bhabhra,, S. Park,, D. S. Perlin,, D. S. Askew, and, J. C. Rhodes. 2005. A fungus-specific Ras homolog contributes to the hyphal growth and virulence of Aspergillus fumigatus. Eukaryot. Cell 4: 1982– 1989.
41. Fournier,, I.,, J. Barwicz, and, P. Tancrede. 1998. The structuring effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers: a differential scanning calorimetry study. Biochim. Biophys. Acta 1373: 76– 86.
42. Gabriel,, I.,, J. Olchowy,, A. Stanislawska-Sachadyn, T. Mio,, J. Kur, and, S. Milewski. 2004. Phosphorylation of glucosamine-6-phosphate synthase is important but not essential for germination and mycelial growth of Candida albicans. FEMS Microbiol. Lett. 235: 73– 80.
43. Gal-Mor, O., and, B. B. Finlay. 2006. Pathogenicity islands: a molecular toolbox for bacterial virulence. Cell. Microbiol. 8: 1707– 1719.
44. Gardner, W. J., and, R. A. Woods. 1979. Isolation and characterisation of guanine auxotrophs in Saccharomyces cerevisiae. Can. J. Micro-biol. 25: 380– 389.
45. Gerbaud,, E.,, F. Tamion,, C. Girault,, K. Clabault,, S. Lepretre,, J. Leroy, and, G. Bonmarchand. 2003. Persistent acute tubular toxicity after switch from conventional amphotericin B to liposomal amphotericin B (Ambisome). J. Antimicrob. Chemother. 51: 473– 475.
46. Giaever,, G.,, A. M. Chu,, L. Ni,, C. Connelly,, L. Riles,, S. Véronneau, S. Dow,, A. Lucau-Danila, K. Anderson,, B. André,, A. P. Arkin,, A. Astromoff,, M. El-Bakkoury,, R. Bangham,, R. Benito,, S. Brachat,, S. Campanaro,, M. Curtiss,, K. Davis,, A. Deutschbauer,, K. D. Entian,, P. Flaherty,, F. Foury,, D. J. Garfinkel,, M. Gerstein,, D. Gotte,, U. Güldener,, J. H. Hegemann,, S. Hempel,, Z. Herman,, D. F. Jaramillo,, D. E. Kelly,, S. L. Kelly,, P. Kötter, D. LaBonte,, D. C. Lamb,, N. Lan,, H. Liang,, H. Liao,, L. Liu,, C. Luo,, M. Lussier,, R. Mao,, P. Menard,, S. L. ooi,, J. L. Revuelta,, C. J. Roberts,, M. Rose,, P. Ross-Macdonald, B. Scherens,, G. Schimmack,, B. Shafer,, D. D. Shoemaker,, S. Sookhai-Mahadeo, R. K. Storms,, J. N. Strathern,, G. Valle,, M. Voet,, G. Volckaert,, C. Y. Wang,, T. R. Ward,, J. Wil-helmy, E. A. Winzeler,, Y. Yang,, G. Yen,, E. Youngman,, K. Yu,, H. Bussey,, J. D. Boeke,, M. Snyder,, P. Philippsen,, R. W. Davis, and, M. Johnston. 2002. Functional profiling of the Saccharomyces cer-evisiae genome. Nature 418: 387– 391.
48. Groll,, A. H., and, T. J. Walsh. 2001. Caspofungin: pharmacology, safety and therapeutic potential in superficial and invasive fungal infections. Expert Opin. Investig. Drugs 10: 1545– 1558.
49. Haselbeck,, R.,, D. Wall,, B. Jiang,, T. Ketela,, J. Zyskind,, H. Bussey,, J. G. Foulkes, and, T. Roemer. 2002. Comprehensive essential gene identification as a platform for novel anti-infective drug discovery. Curr. Pharm. Des. 8: 1155– 1172.
50. Hashida-Okado, T.,, A. Ogawa,, M. Endo,, R. Yasumoto,, K. Takesako, and, I. Kato. 1996. AUR1, a novel gene conferring aureobasidin resistance on Saccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells. Mol. Gen. Genet. 251: 236– 244.
51. Heidler,, S. A., and, J. A. Radding. 1995. The AUR1 gene in Sacchar-omyces cerevisiae encodes dominant resistance to the antifungal agent aureobasidin A (LY295337). Antimicrob. Agents Chemother. 39: 2765– 2769.
52. Heidler,, S. A., and, J. A. Radding. 2000. Inositol phosphoryl transferases from human pathogenic fungi. Biochim. Biophys. Acta 1500: 147– 152.
53. Heidtman,, M.,, C. Z. Chen,, R. N. Collins, and, C. Barlowe. 2003. A role for Yip1p in COPII vesicle biogenesis. J. Cell Biol. 163: 57– 69.
54. Henry,, C.,, I. Mouyna, and, J. P. Latgé. 2007. Testing the efficacy of RNA interference constructs in Aspergillus fumigatus. Curr. Genet. 51: 277– 284.
55. Hirt,, R. P.,, S. Müller, T. M. Embley, and, G. H. Coombs. 2002. The diversity and evolution of thioredoxin reductase: new perspectives. Trends Parasitol. 18: 302– 308.
56. Hope,, W. W.,, S. Shoham, and, T. J. Walsh. 2007. The pharmacology and clinical use of caspofungin. Expert Opin. Drug Metab. Toxicol. 3: 263– 274.
57. Hu,, W., S. Sillaots,, S. Lemieux,, J. Davison,, S. Kauffman,, A. Breton,, A. Linteau,, C. Xin,, J. Bowman,, J. Becker,, B. Jiang, and, T. Roemer. 2007. Essential gene identification and drug target prioritization in Aspergillus fumigatus. PLoS Pathog. 3: e24.
58. Jiang, B.,, H. Bussey, and, T. Roemer. 2002. Novel strategies in antifungal lead discovery. Curr. Opin. Microbiol. 5: 466– 471.
59. Jones,, T.,, N. A. Federspiel,, H. Chibana,, J. Dungan,, S. Kalman,, B. B. Magee,, G. Newport,, Y. R. Thorstenson,, N. Agabian,, P. T. Magee,, R. W. Davis, and, S. Scherer. 2004. The diploid genome sequence of Candida albicans. Proc. Natl. Acad. Sci. USA 101: 7329– 7334.
60. Kalb,, V. F.,, C. W. Woods,, T. G. Turi,, C. R. Dey,, T. R. Sutter, and, J. C. Loper. 1987. Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA 6: 529– 557.
61. Kauffman,, C. A. 2006. Clinical efficacy of new antifungal agents. Curr. Opin. Microbiol. 9: 483– 488.
62. Khalaj,, V.,, H. Eslami,, M. Azizi,, N. Rovira-Graells, and, M. Bromley. 2007. Efficient downregulation of albl gene using an AMA1 -based episomal expression of RNAi construct in Aspergillus fumigatus. FEMS Microbiol. Lett. 270: 250– 254.
63. Kirsch,, D. R., and, R. R. Whitney. 1991. Pathogenicity of Candida albicans auxotrophic mutants in experimental infections. Infect. Im-mun. 59: 3297– 3300.
64. Krappmann,, S.,, C. Sasse, and, G. H. Braus. 2006. Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joining-deficient genetic background. Eukar-yot. Cell 5: 212– 215.
65. Kupfahl, C, T. Heinekamp,, G. Geginat,, T. Ruppert,, A. Hard,, H. Hof, and, A. A. Brakhage. 2006. Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model. Mol. Microbiol. 62: 292– 302.
66. Lagorce,, A.,, V. Le Berre-Anton, B. Aguilar-Uscanga, H. Martin-Yken, A. Dagkessamanskaia, and, J. Frangois. 2002. Involvement of GFA1, which encodes glutamine-fructose-6-phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell-wall defects in Saccharomyces cerevisiae. Eur. J. Biochem. 269: 1697– 1707.
67. Lamarre, C, O., Ibrahim-Granet, C., Du,, R. Calderone, and, J. P. Latge. 2007. Characterization of the SKN7 ortholog of Aspergillus fumigatus. Fungal Genet. Biol. 44: 682– 690.
68. Latgé,, J. P. 1999. Aspergillus fumigatus and aspergillosis. Clin. Microbiol. Rev. 12: 310– 350.
69. Latgé,, J. P. 2001. The pathobiology of Aspergillus fumigatus. Trends Microbiol. 9: 382– 389.
70. Liebmann,, B.,, T. W. Mühleisen,, M. Müller,, M. Hecht,, G. Weidner,, A. Braun,, M. Brock, and, A. A. Brakhage. 2004. Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis. Arch. Microbiol. 181: 378– 383.
71. Liu,, H.,, T. R. Cottrell,, L. M. Pierini,, W. E. Goldman, and, T. L. Doering. 2002. RNA interference in the pathogenic fungus Cryp-tococcus neoformans. Genetics 160: 463– 470.
72. Lupetti,, A.,, R. Danesi,, M. Campa,, M. Del Tacca, and, S. Kelly. 2002. Molecular basis of resistance to azole antifungals. Trends Mol. Med. 8: 76– 81.
73. Mandala,, S. M.,, R. A. Thornton,, M. Rosenbach,, J. Milligan,, M. Garcia-Calvo, H. G. Bull, and, M. B. Kurtz. 1997. Khafrefungin, a novel inhibitor of sphingolipid synthesis. J. Biol. Chem. 272: 32709– 32714.
74. Mandala,, S. M.,, R. A. Thornton,, J. Milligan,, M. Rosenbach,, M. Garcia-Calvo, H. G. Bull,, G. Harris,, G. K. Abruzzo,, A. M. Flattery, C. J. Gill,, K. Bartizal,, S. Dreikorn, and, M. B. Kurtz. 1998. Rust-micin, a potent antifungal agent, inhibits sphingolipid synthesis at inositol phosphoceramide synthase. Biol. Chem. 273: 14942– 14949.
75. Mann,, P. A.,, R. M. Parmegiani,, S. Q. Wei,, C. A. Mendrick,, X. Li,, D. Loebenberg,, B. DiDomenico,, R. S. Hare,, S. S. Walker, and, P. M. McNicholas. 2003. Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P450 14a-demethylase. Anti-microb. Agents Chemother. 47: 577– 581.
76. Marr,, K. A.,, R. A. Carter,, F. Crippa,, A. Wald, and, L. Corey. 2002. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin. Infect. Dis. 34: 909– 917.
77. McNeil,, M. M.,, S. L. Nash,, R. A. Hajjeh,, M. A. Phelan,, L. A. Conn,, B. D. Plikaytis, and, D. W. Warnock. 2001. Trends in mortality due to invasive mycotic diseases in the United States, 1980-1997. Clin. Infect. Dis. 33: 641– 647.
78. Mellado,, E.,, T. M. Diaz-Guerra, M. Cuenca-Estrella, and, J. L. Rodriguez-Tudela. 2001. Identification of two different 14-a sterol demethylaserelated genes (cypSIA and cypSIB) in Aspergillus fumigatus and other Aspergillus species. J. Clin. Microbiol. 39: 2431– 2438.
79. Missall,, T. A., and, J. K. Lodge. 2005. Thioredoxin reductase is essential for viability in the fungal pathogen Cryptococcus neoformans. Eukaryot. Cell 4: 487– 489.
80. Mnaimneh,, S.,, A. P. Davierwala,, J. Haynes,, J. Moffat,, W. T. Peng,, W. Zhang,, X. Yang,, J. Pootoolal,, G. Chua,, A. Lopez,, M. Troches-set, D. Morse,, N. J. Krogan,, S. L. Hiley,, Z. Li,, Q. Morris,, J. Grigull,, N. Mitsakakis,, C. J. Roberts, J, F. Greenblatt,, C. Boone,, C. A. Kaiser,, B. J. Andrews, and, T. R. Hughes. 2004. Exploration of essential gene functions via titratable promoter alleles. Cell 118: 31– 44.
81. Model,, K.,, T. Prinz,, T. Ruiz,, M. Radermacher,, T. Krimmer,, W. Kühl-brandt, N. Pfanner, and, C. Meisinger. 2002. Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex. J. Mol. Biol. 316: 657– 666.
82. Mouyna,, I.,, C. Henry,, T. L. Doering, and, J. P. Latgé. 2004. Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol. Lett. 237: 317– 324.
83. Muro-Pastor, M. I.,, R. Gonzalez,, J. Strauss,, F. Narendja, and, C. Scazzocchio. 1999. The GATA factor AreA is essential for chromatin remodelling in a eukaryotic bidirectional promoter. EMBO J. 18: 1584– 1597.
84. Nagiec,, M. M.,, E. E. Nagiec,, J. A. Baltisberger,, G. B. Wells,, R. L. Lester, and, R. C. Dickson. 1997. Sphingolipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cer-evisiae by the AUR1 gene. J. Biol. Chem. 272: 9809– 9817.
85. Nargang,, F. E.,, K. P. Künkele,, A. Mayer,, R. G. Ritzel,, W. Neupert, and, R. Lill. 1995. ’Sheltered disruption’ of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex. EMBO J. 14: 1099– 1108.
86. Nierman,, W.C.,, A. Pain,, M. J. Anderson,, J. R. Wortman,, H. S. Kim,, J. Arroyo,, M. Berriman,, K. Abe,, D. B. Archer,, C. Bermejo,, J. Bennett,, P. Bowyer,, D. Chen,, M. Collins,, R. Coulsen,, R. Davies,, P. S. Dyer,, M. Farman,, N. Fedorova,, N. Fedorova,, T. V. Feldblyum,, R. Fischer,, N. Fosker,, A. Fraser,, J. L. Garcia,, M. J. Garcia,, A. Goble,, G. H. Goldman,, K. Gomi,, S. Griffith-Jones, R. Gwilliam,, B. Haas,, H. Haas,, D. Harris,, H. Horiuchi,, J. Huang,, S. Humphray,, J. Jimenez,, N. Keller,, H. Khouri,, K. Kitamoto,, T. Kobayashi,, S. Kon-zack, R. Kulkarni,, T. Kumagai,, A. Lafon,, J. P. Large,, W. Li,, A. Lord,, C. Lu,, W. H. Majoros,, G. S. May,, B. L. Miller,, Y. Mohamoud, M. Molina,, M. Monod,, I. Mouyna,, S. Mulligan,, L. Murphy,, S. O’Neil, I. Paulsen,, M. A. Penalva,, M. Pertea,, C. Price,, B. L. Pritchard,, M. A. Quail,, E. Rabbinowitsch,, N. Rawlins,, M. A. Ra-jandream, U. Reichard,, H. Renauld,, G. D. Robson,, S. Rodriguez de Cordoba, J. M. Rodriguez-Pena, C. M. Ronning,, S. Rutter,, S. L. Salzberg,, M. Sanchez,, J. C. Sanchez-Ferrero, D. Saunders,, K. Seeger,, R. Squares,, S. Squares,, M. Takeuchi,, F. Tekaia,, G. Turner,, C. R. Vazquez de Aldana,, J. Weidman,, O. White,, J. Woodward,, J. H. Yu,, V. Fraser,, J. E. Galagan,, K. Asai,, M. Machida,, N. Hall,, B. Barrell, and, D. W. Denning. 2005. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438: 1151– 1156.
87. Ninomiya,, Y.,, K. Suzuki,, C. Ishii, and, H. Inoue. 2004. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc. Natl. Acad. Sci. USA 101: 12248– 12253.
88. Odds,, F. C. 2005. Genomics, molecular targets and the discovery of antifungal drugs. Rev. Iberoam. Micol. 22: 229– 237.
89. Oliveira,, M. A.,, K. F. Discola,, S. V. Alves,, J. A. Barbosa,, F. J. Med-rano,, L. E. Netto, and, N. G. Guimarães. 2005. Crystallization and preliminary X-ray diffraction analysis of NADPH-dependent thioredoxin reductase I from Saccharomyces cerevisiae. Acta Crystallogr. F 61: 387– 390.
90. Osmani,, A. H.,, J. Davies,, H. L. Liu,, A. Nile, and, S. A. Osmani. 2004. Systematic deletion and mitotic localization of the nuclear pore complex proteins of Aspergillus nidulans. Mol. Biol. Cell 17: 4946– 4961.
91. Osmani,, A. H.,, B. R. Oakley, and, S. A. Osmani. 2006. Identification and analysis of essential Aspergillus nidulans genes using the heterokaryon rescue technique. Nat. Protoc. 1: 2517– 2526.
92. Osmani,, S. A.,, D. B. Engle,, J. H. Doonan, and, N. R. Morris. 1988. Spindle formation and chromatin condensation in cells blocked at interphase by mutation of a negative cell cycle control gene. Cell 52: 241– 251.
93. Pathak,, A.,, F. D. Pien, and, L. Carvalho. 1998. Amphotericin B use in a community hospital, with special emphasis on side effects. Clin. Infect. Dis. 26: 334– 338.
94. Perfect,, J. R. 1996. Fungal virulence genes as targets for antifungal chemotherapy. Antimicrob. Agents Chemother. 40: 1577– 1583.
95. Perlin,, D. S. 2007. Resistance to echinocandin-class antifungal drugs. Drug Resist. Update 10: 121– 130.
96. Pontecorvo,, G.,, J. A. Roper, and, E. Forbes. 1953. Genetic recombination without sexual reproduction in Aspergillus niger. J. Gen. Microbiol. 8: 198– 210.
97. Punt,, P. J.,, J. Strauss,, R. Smit,, J. R. Kinghorn,, C. A. van den Hondel, and, C. Scazzocchio. 1995. The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally. Mol. Cell. Biol. 15: 5688– 5699.
98. Rapaport,, D. 2005. How does the TOM complex mediate insertion of precursor proteins into the mitochondrial outer membrane? J. Cell Biol. 17: 419– 423.
99. Rementeria,, A.,, N. López-Molina,, A. Ludwig,, A. B. Vivanco,, J. Bi-kandi,, J. Pontón, and, J. Garaizar. 2005. Genes and molecules involved in Aspergillus fumigatus virulence. Rev. Iberoam. Micol. 22: 1– 23.
100. Rodriguez-Suarez, R.,, D. Xu,, K. Veillette,, J. Davison,, S. Sillaots,, S. Kauffman,, W. Hu,, J. Bowman,, N. Martel,, S. Trosok,, H. Wang,, L. Zhang,, L. Y. Huang,, Y. Li,, F. Rahkhoodaee,, T. Ransom,, D. Gau-vin,, C. Douglas,, P. Youngman,, J. Becker,, B. Jiang, and, T. Roemer. 2007. Mechanism-of-action determination of GMP synthase inhibitors and target validation in Candida albicans and Aspergillus fumigatus. Chem. Biol. 14: 1163– 1175.
101. Roemer,, T.,, B. Jiang,, J. Davison,, T. Ketela,, K. Veillette,, A. Breton,, F. Tandia,, A. Linteau,, S. Sillaots,, C. Marta,, N. Martel,, S. Veron-neau, S. Lemieux,, S. Kauffman,, J. Becker,, R. Storms,, C. Boone, and, H. Bussey. 2003. Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery. Mol. Microbiol. 50: 167– 181.
102. Romano,, J.,, G. Nimrod,, N. Ben-Tal, Y. Shadkchan,, K. Baruch,, H. Sharon, and, N. Osherov. 2006. Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence. Microbiology 152: 1919– 1928.
103. Romero,, B.,, G. Turner,, I. Olivas,, F. Laborda, and, J. R. De Lucas. 2003. The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen. Fungal Genet. Biol. 40: 103– 114.
104. Ronning,, C. M.,, N. D. Fedorova,, P. Bowyer,, R. Coulson,, G. Goldman,, H. S. Kim,, G. Turner,, J. R. Wortman,, J. Yu,, M. J. Anderson,, D. W. Denning, and, W. C. Nierman. 2005. Genomics of Aspergillus fumigatus. Rev. Iberoam. Micol. 22: 223– 228.
105. Ross-Macdonald, P.,, P. S. Coelho,, T. Roemer,, S. Agarwal,, A. Kumar,, R. Jansen,, K. H. Cheung,, A. Sheehan,, D. Symoniatis,, L. Umansky,, M. Heidtman,, F. K. Nelson,, H. Iwasaki,, K. Hager,, M. Gerstein,, P. Miller,, G. S. Roeder, and, M. Snyder. 1999. Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Nature 402: 413– 418.
106. Salama,, N. R.,, J. S. Chuang, and, R. W. Schekman. 1997. Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum. Mol. Biol. Cell 8: 205– 217.
107. Schrettl,, M.,, E. Bignell,, C. Kragl,, C. Joechl,, T. Rogers,, H. N. Arst, Jr., K. Haynes, and, H. Haas. 2004. Siderophore biosynthesis but not reductive iron assimilation is essential for Aspergillus fumigatus virulence. J. Exp. Med. 200: 1213– 1219.
108. Sheppard,, D. C.,, T. Doedt,, L. Y. Chiang,, H. S. Kim,, D. Chen,, W. C. Nierman, and, S. G. Filler. 2005. The Aspergillus fumigatus StuA protein governs the up-regulation of a discrete transcriptional program during the acquisition of developmental competence. Mol. Biol. Cell 16: 5866– 5879.
109. Sherman,, E. L.,, R. D. Taylor,, N. E. Go, and, F. E. Nargang. 2006. Effect of mutations in Tom40 on stability of the translocase of the outer mitochondrial membrane (TOM) complex, assembly of Tom40, and import of mitochondrial preproteins. J. Biol. Chem. 281: 22554– 22565.
110. Smith,, R. J.,, S. Milewski,, A. J. Brown, and, G. W. Gooday. 1996. Isolation and characterization of the GFA1 gene encoding the glu-tamine: fructose-6-phosphate amidotransferase of Candida albicans. J. Bacteriol. 178: 2320– 2327.
111. Smith,, V.,, K. N. Chou,, D. Lashkari,, D. Botstein, and, P. O. Brown. 1996. Functional analysis of the genes of yeast chromosome V by genetic footprinting. Science 274: 2069– 2074.
112. Som,, T., and, V. S. Kolaparthi. 1994. Developmental decisions in As-pergillus nidulans are modulated by Ras activity. Mol. Cell. Biol. 14: 5333– 5348.
113. South,, S. T.,, K. A. Sacksteder,, X. Li,, Y. Liu, and, S. J. Gould. 2000. Inhibitors of COPI and COPII do not block PEX3-mediated peroxisome synthesis. J. Cell Biol. 149: 1345– 1360.
114. Spanakis,, E. K.,, G. Aperis, and, E. Mylonakis. 2006. New agents for the treatment of fungal infections: clinical efficacy and gaps in coverage. Clin. Infect. Dis. 43: 1060– 1068.
115. Steinbach,, W. J.,, R. A. Cramer, Jr., B. Z. Perfect,, Y. G. Asfaw,, T. C. Sauer,, L. K. Najvar,, W. R. Kirkpatrick,, T. F. Patterson,, D. K. Benjamin, Jr., J. Heitman, and, J. R. Perfect. 2006. Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus. Eukaryot. Cell 5: 1091– 1103.
116. Stromnaes,, O., and, E. D. Garber. 1963. Heterocaryosis and the parasexual cycle in Aspergillus fumigatus. Genetics 48: 653– 662.
117. Sugimoto,, Y.,, H. Sakoh, and, K. Yamada. 2004. IPC synthase as a useful target for antifungal drugs. Curr. Drug Targets Infect. Disord. 4: 311– 322.
118. Takatsu,, T.,, H. Nakayama,, A. Shimazu,, K. Furihata,, K. Ikeda,, K. Furihata,, H. Seto, and, N. Otake. 1985. Rustmicin, a new macrolide antibiotic active against wheat stem rust fungus. J. Antibiot. (Tokyo) 38: 1806– 1809.
119. Takesako,, K.,, K. Ikai,, F. Haruna,, M. Endo,, K. Shimanaka,, E. Sono,, T. Nakamura,, I. Kato, and, H. Yamaguchi. 1991. Aureobasidins, new antifungal antibiotics. Taxonomy, fermentation, isolation, and properties. J. Antibiot. (Tokyo) 44: 919– 924.
120. Takesako,, K.,, H. Kuroda,, T. Inoue,, F. Haruna,, Y. Yoshikawa,, I. Kato,, K. Uchida,, T. Hiratani, and, H. Yamaguchi. 1993. Biological properties of aureobasidin A, a cyclic depsipeptide antifungal antibiotic. J. Antibiot. (Tokyo) 46: 1414– 1420.
121. Taylor,, R. D.,, B. J. McHale, and, F. E. Nargang. 2003. Characterization of Neurospora crassa Tom40-deficient mutants and effect of specific mutations on Tom40 assembly. J. Biol. Chem. 278: 765– 775.
122. Tekaia,, F., and, J. P. Latge. 2005. Aspergillus fumigatus: saprophyte or pathogen? Curr. Opin. Microbiol. 8: 385– 392.
123. Teplyakov,, A.,, G. Obmolova,, M. A. Badet-Denisot, and, B. Badet. 1999. The mechanism of sugar phosphate isomerization by glucosamine 6-phosphate synthase. Protein Sci. 8: 596– 602.
124. Timberlake,, W. E., and, M. A. Marshall. 1988. Genetic regulation of development in Aspergillus nidulans. Trends Genet. 4: 162– 169.
125. Tsai,, H. F.,, M. H. Wheeler,, Y. C. Chang, and, K. J. Kwon-Chung. 1999. A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus. J. Bacteriol. 181: 6469– 6477.
126. Uhl,, M. A.,, M. Biery,, N. Craig, and, A. D. Johnson. 2003. Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen C. albicans. EMBO J. 22: 2668– 2678.
127. Vogt,, K.,, R. Bhabhra,, J. C. Rhodes, and, D. S. Askew. 2005. Doxycycline-regulated gene expression in the opportunistic fungal pathogen Aspergillus fumigatus. BMC Microbiol. 5: 1.
128. Watson,, J. M.,, A. F. Fusaro,, M. Wang, and, P. M. Waterhouse. 2005. RNA silencing platforms in plants. FEBS Lett. 579: 5982– 5987.
129. Winzeler,, E. A.,, D. D. Shoemaker,, A. Astromoff,, H. Liang,, K. Anderson,, B. Andre,, R. Bangham,, R. Benito,, J. D. Boeke,, H. Bussey,, A. M. Chu,, C. Connelly,, K. Davis,, F. Dietrich,, S. W. Dow,, M. El Bakkoury, F. Foury,, S. H. Friend,, E. Gentalen,, G. Giaever,, J. H. Hegemann,, T. Jones,, M. Laub,, H. Liao,, N. Liebundguth,, D. J. Lockhart,, A. Lucau-Danila, M. Lussier,, N. M’Rabet, P. Menard,, M. Mittmann,, C. Pai,, C. Rebischung,, J. L. Revuelta,, L. Riles,, C. J. Roberts,, P. Ross-MacDonald, B. Scherens,, M. Snyder,, S. Sookhai-Mahadeo, R. K. Storms,, S. Véronneau,, M. Voet,, G. Volckaert,, T. R. Ward,, R. Wysocki,, G. S. Yen,, K. Yu,, K. Zimmermann,, P. Philippsen,, M. Johnston, and, R. W. Davis. 1999. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285: 901– 906.
130. Yamasaki,, A.,, K. Tani,, A. Yamamoto,, N. Kitamura, and, M. Komada. 2006. The Ca 2+-binding protein ALG-2 is recruited to endoplasmic reticulum exit sites by Sec31A and stabilizes the localization of Sec31A. Mol. Biol. Cell 17: 4876– 4887.
131. Zamore,, P. D., and, N. Aronin. 2003. siRNAs knock down hepatitis. Nat. Med. 9: 266– 267.

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