1887

Chapter 43 : Genetic and Proteomic Analysis of Fungal Virulence

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $15.00

Preview this chapter:
Zoom in
Zoomout

Genetic and Proteomic Analysis of Fungal Virulence, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555815776/9781555813680_Chap43-1.gif /docserver/preview/fulltext/10.1128/9781555815776/9781555813680_Chap43-2.gif

Abstract:

This chapter explores the genetic and proteomic approaches that are now feasible for many fungal systems. Post-genomic approaches to the analysis of biological function, networks, and processes often include techniques that permit global analysis of gene expression at the protein level. Analysis of protein expression during modulation of external conditions or during particular developmental states can provide useful clues about which genes might be important for a particular function. These proteomic approaches are complementary to techniques, such as serial analysis of gene expression (SAGE) and microarrays, that measure gene expression at the RNA level. Signature-tagged mutagenesis (STM) was also used to identify var. mutants with altered virulence in a mouse model of disseminated disease. Restriction enzyme-mediated insertion has also been used with fungi to promote relatively random insertions in fungal genomes. It has been commonly used in phytopathogens, but the only published studies in a human pathogen are those done with and . Proteomic analysis of the response to several antifungal drugs showed that inhibition of specific targets resulted in a unique profile. Mechanisms of antifungal resistance can be elucidated by identification of proteins induced by treatment with antifungal agents. To determine mechanisms of fungal virulence, it is important to study the expression of proteins during the interaction of a pathogen with host cells, either in vitro or in vivo.

Citation: Lodge J, Lorenz M. 2006. Genetic and Proteomic Analysis of Fungal Virulence, p 643-655. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch43

Key Concept Ranking

Cell Wall Biosynthesis
0.4323581
Sodium Dodecyl Sulfate
0.4323581
DNA Restriction Enzymes
0.42173293
0.4323581
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of Figure 1
Figure 1

Schematic for STM. A unique signature tag (ST) oligonucleotide is synthesized, incorporated into a plasmid vector, and flanked by primer binding sites. Each “tagged” vector is used to transform cells producing many uniquely tagged mutants. One mutant representing each uniquely tagged vector (up to 96 in total) is assembled into an input pool. Tags present in the input pool are PCR amplified, labeled, and used to probe a filter containing dot blots of each tag forming the preinoculum filter. The pooled organisms are inoculated into an animal, and the infection is allowed to proceed for a determined period. Organisms are recovered from tissue at the site of infection, and the tags are amplified using common primers, labeled, and used to probe a duplicate filter (postinoculum filter). Tags missing from the pool are identified by a loss of signal on the blot. Mutants that overproliferate can also be identified by having a much stronger signal compared to the input blot. Mutants that did not have a change in their virulence have a similar signal to that of the input blot.

Citation: Lodge J, Lorenz M. 2006. Genetic and Proteomic Analysis of Fungal Virulence, p 643-655. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch43
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of Figure 2
Figure 2

Comparison of lysates from grown at 25 and 37°C by 2D gel electrophoresis. The two thiol peroxidases, Tsa1 and Tsa3, that are up-regulated at higher temperature are indicated by arrows. Reprinted from reference .

Citation: Lodge J, Lorenz M. 2006. Genetic and Proteomic Analysis of Fungal Virulence, p 643-655. In Heitman J, Filler S, Edwards, Jr. J, Mitchell A (ed), Molecular Principles of Fungal Pathogenesis. ASM Press, Washington, DC. doi: 10.1128/9781555815776.ch43
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555815776.ch43
1. Aggarwal, K., and, K. H. Lee. 2003. Functional genomics and proteomics as a foundation for systems biology. Brief Funct. Genomic Proteomic 2:175184.
2. Alani, E.,, L. Cao, and, N. Kleckner. 1987. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116:541545.
3. Bader, G. D.,, A. Heilbut,, B. Andrews,, M. Tyers,, T. Hughes, and, C. Boone. 2003. Functional genomics and proteomics: charting a multidimensional map of the yeast cell. Trends Cell Biol. 13:344356.
4. Brown, D. H.,, I. V. Slobodkin, and, C. A. Kumamoto. 1996. Stable transformation and regulated expression of an inducible reporter construct in Candida albicans using restriction enzyme-mediated integration. Mol. Gen. Genet. 251:7580.
5. Brown, J. S.,, A. Aufauvre-Brown, and, D. W. Holden. 1998. Insertional mutagenesis of Aspergillus fumigatus. Mol. Gen. Genet. 259:327335.
6. 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. 36:13711380.
7. Bruneau, J. M.,, T. Magnin,, E. Tagat,, R. Legrand,, M. Bernard,, M. Diaquin,, C. Fudali, and, J. P. Latge. 2001. Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis. Electrophoresis 22:28122823.
8. Bruneau, J. M.,, I. Maillet,, E. Tagat,, R. Legrand,, F. Supatto,, C. Fudali,, J. P. Caer,, V. Labas,, D. Lecaque, and, J. Hodgson. 2003. Drug induced proteome changes in Candida albicans: comparison of the effect of β(1,3)-glucan synthase inhibitors and two triazoles, fluconazole and itraconazole. Proteomics 3:325336.
9. Bruno, V. M., and, A. P. Mitchell. 2004. Large-scale gene function analysis in Candida albicans. Trends Microbiol. 12:157161.
10. Carpenter, A. E., and, D. M. Sabatini. 2004. Systematic genome-wide screens of gene function. Nat. Rev. Genet. 5:1122.
11. Cheng, S.,, M. H. Nguyen,, Z. Zhang,, H. Jia,, M. Handfield, and, C. J. Clancy. 2003. Evaluation of the roles of four Candida albicans genes in virulence by using gene disruption strains that express URA3 from the native locus. Infect. Immun. 71:61016103.
12. Choi, W.,, Y. J. Yoo,, M. Kim,, D. Shin,, H. B. Jeon, and, W. Choi. 2003. Identification of proteins highly expressed in the hyphae of Candida albicans by two-dimensional electrophoresis. Yeast 20:10531060.
13. Conrads, T. P.,, G. A. Anderson,, T. D. Veenstra,, L. PasaTolic, and, R. D. Smith. 2000. Utility of accurate mass tags for proteome-wide protein identification. Anal. Chem. 72:33493354.
14. Cormack, B. P.,, N. Ghori, and, S. Falkow. 1999. An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Science 285:578582.
15. Davidson, R. C.,, J. R. Blankenship,, P. R. Kraus,, M. de Jesus Berrios,, C. M. Hull,, C. D’Souza,, P. Wang, and, J. Heitman. 2002. A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148:26072615.
16. Davidson, R. C.,, T. D. Moore,, A. R. Odom, and, J. Heitman. 2000. Characterization of the MFα pheromone of the human fungal pathogen Cryptococcus neoformans. Mol. Microbiol. 38:10171026.
17. Davis, D. A.,, V. M. Bruno,, L. Loza,, S. G. Filler, and, A. P. Mitchell. 2002. Candida albicans Mds3p, a conserved regulator of pH responses and virulence identified through insertional mutagenesis. Genetics 162:15731581.
18. 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. 19:235241.
19. de Groot, P. W.,, A. D. de Boer,, J. Cunningham,, H. L. Dekker,, L. de Jong,, K. J. Hellingwerf,, C. de Koster, and, F. M. Klis. 2004. Proteomic analysis of Candida albicans cell walls reveals covalently bound carbohydrate-active enzymes and adhesins. Eukaryot. Cell 3:955965.
20. Del Poeta, M.,, D. L. Toffaletti,, T. H. Rude,, C. C. Dykstra,, J. Heitman, and, J. R. Perfect. 1999. Topoisomerase I is essential in Cryptococcus neoformans: role in pathobiology and as an antifungal target. Genetics 152:167178.
21. Fox, D. S.,, M. C. Cruz,, R. A. Sia,, H. Ke,, G. M. Cox,, M. E. Cardenas, and, J. Heitman. 2001. Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans. Mol. Microbiol. 39:835849.
22. Ghaemmaghami, S.,, W. K. Huh,, K. Bower,, R. W. Howson,, A. Belle,, N. Dephoure,, E. K. O’Shea, and, J. S. Weissman. 2003. Global analysis of protein expression in yeast. Nature 425:737741.
23. Giaever, G.,, A. M. Chu,, L. Ni,, C. Connelly,, L. Riles, et al. 2002. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387391.
24. Gorlach, J. M.,, H. C. McDade,, J. R. Perfect, and, G. M. Cox. 2002. Antisense repression in Cryptococcus neofor-mans as a laboratory tool and potential antifungal strategy. Microbiology 148:213219.
25. Graves, P. R., and, T. A. Haystead. 2003. A functional proteomics approach to signal transduction. Recent Prog. Horm. Res. 58:124.
26. Gygi, S. P.,, B. Rist,, S. A. Gerber,, F. Turecek,, M. H. Gelb, and, R. Aebersold. 1999. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17:994999.
27. Gygi, S. P.,, Y. Rochon,, B. R. Franza, and, R. Aebersold. 1999. Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 19:17201730.
28. Hensel, M.,, J. E. Shea,, C. Gleeson,, M. D. Jones,, E. Dalton, and, D. W. Holden. 1995. Simultaneous identification of bacterial virulence genes by negative selection. Science 269:400403.
29. Hernandez, R.,, C. Nombela,, R. Diez-Orejas, and, C. Gil. 2004. Two-dimensional reference map of Candida albi-cans hyphal forms. Proteomics 4:374382.
30. Ho, Y.,, A. Gruhler,, A. Heilbut,, G. D. Bader,, L. Moore,, S. L. Adams,, A. Millar,, P. Taylor,, K. Bennett,, K. Boutilier,, L. Yang,, C. Wolting,, I. Donaldson,, S. Schandorff,, J. Shewnarane,, M. Vo,, J. Taggart,, M. Goudreault,, B. Muskat,, C. Alfarano,, D. Dewar,, Z. Lin,, K. Michalickova,, A. R. Willems,, H. Sassi,, P. A. Nielsen,, K. J. Rasmussen,, J. R. Andersen,, L. E. Johansen,, L. H. Hansen,, H. Jespersen,, A. Podtelejnikov,, E. Nielsen,, J. Crawford,, V. Poulsen,, B. D. Sorensen,, J. Matthiesen,, R. C. Hendrickson,, F. Gleeson,, T. Pawson,, M. F. Moran,, D. Durocher,, M. Mann,, C. W. Hogue,, D. Figeys, and, M. Tyers. 2002. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415:180183.
31. Huh, W. K.,, J. V. Falvo,, L. C. Gerke,, A. S. Carroll,, R. W. Howson,, J. S. Weissman, and, E. K. O’Shea. 2003. Global analysis of protein localization in budding yeast. Nature 425:686691.
32. Hutvagner, G., and, P. D. Zamore. 2002. RNAi: nature abhors a double-strand. Curr. Opin. Genet. Dev. 12:225232.
33. Ideker, T.,, T. Galitski, and, L. Hood. 2001. A new approach to decoding life: systems biology. Annu. Rev. Genomics Hum. Genet. 2:343372.
34. Idnurm, A.,, J. L. Reedy,, J. C. Nussbaum, and, J. Heitman. 2004. Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot. Cell 3:420429.
35. Li, M.,, S. J. Martin,, V. M. Bruno,, A. P. Mitchell, and, D. A. Davis. 2004. Candida albicans Rim13p, a protease required for Rim101p processing at acidic and alkaline pHs. Eukaryot. Cell 3:741751.
36. Liu, H.,, T. R. Cottrell,, L. M. Pierini,, W. E. Goldman, and, T. L. Doering. 2002. RNA interference in the pathogenic fungus Cryptococcus neoformans. Genetics 160:463470.
37. MacKay, V. L.,, X. Li,, M. R. Flory,, E. Turcott,, G. L. Law,, K. A. Serikawa,, X. L. Xu,, H. Lee,, D. R. Goodlett,, R. Aebersold,, L. P. Zhao, and, D. R. Morris. 2004. Gene expression analyzed by high-resolution state array analysis and quantitative proteomics: response of yeast to mating pheromone. Mol. Cell. Proteomics 3:478489.
38. Martinez-Lopez, R.,, L. Monteoliva,, R. Diez-Orejas,, C. Nombela, and, C. Gil. 2004. The GPI-anchored protein CaEcm33p is required for cell wall integrity, morphogenesis and virulence in Candida albicans. Microbiology 150:33413354.
39. Missall, T. A.,, M. E. Pusateri, and, J. K. Lodge. 2004. Thiol peroxidase is critical for virulence and resistance to nitric oxide and peroxide in the fungal pathogen, Cryptococcus neoformans. Mol. Microbiol. 51:14471458.
40. Missall, T. A., and, J. K. Lodge. 2005. Thioredoxin reductase is essential for viability in the fungal pathogen, Cryptococcus neoformans. Eukaryot. Cell 4:487489.
41. Mouyna, I.,, C. Henry,, T. L. Doering, and, J. P. Latge. 2004. Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol. Lett. 237:317324.
42. Nakayama, H.,, T. Mio,, S. Nagahashi,, M. Kokado,, M. Arisawa, and, Y. Aoki. 2000. Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans. Infect. Immun. 68:67126719.
43. Nelson, R. T.,, J. Hua,, B. Pryor, and, J. K. Lodge. 2001. Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis. Genetics 157:935947.
44. Nelson, R. T.,, B. A. Pryor, and, J. K. Lodge. 2003. Sequence length required for homologous recombination in Cryptococcus neoformans. Fungal Genet. Biol. 38:19.
45. Ory, J. J.,, C. L. Griffith, and, T. L. Doering. 2004. An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter. Yeast 21:919926.
46. Panse, V. G.,, U. Hardeland,, T. Werner,, B. Kuster, and, E. Hurt. 2004. A proteome-wide approach identifies sumoylated substrate proteins in yeast. J. Biol. Chem. 279:4134641351.
47. Pitarch, A.,, M. Sanchez,, C. Nombela, and, C. Gil. 2002. Sequential fractionation and two-dimensional gel analysis unravels the complexity of the dimorphic fungus Candida albicans cell wall proteome. Mol. Cell. Proteomics 1:967982.
48. Pitarch, A.,, M. Sanchez,, C. Nombela, and, C. Gil. 2003. Analysis of the Candida albicans proteome. I. Strategies and applications. J. Chromatogr. Ser. B. 787:101128.
49. Rappleye, C. A.,, J. T. Engle, and, W. E. Goldman. 2004. RNA interference in Histoplasma capsulatum demonstrates a role for α-(1,3)-glucan in virulence. Mol. Microbiol. 53:153165.
50. Roemer, T.,, B. Jiang,, J. Davison,, T. Ketela,, K. Veillette,, A. Breton,, F. Tandia,, A. Linteau,, S. Sillaots,, C. Marta,, N. Martel,, S. Veronneau,, S. Lemieux,, S. Kauffman,, J. Becker,, R. Storms,, C. Boone, and, H. Bussey. 2003. Large-scale essential gene identification in Candida albi-cans and applications to antifungal drug discovery. Mol. Microbiol. 50:167181.
51. Salas, S. D.,, J. E. Bennett,, K. J. Kwon-Chung,, J. R. Perfect, and, P. R. Williamson. 1996. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neofor-mans. J. Exp. Med. 184:377386.
52. Saville, S. P.,, A. L. Lazzell,, C. Monteagudo, and, J. L. Lopez-Ribot. 2003. Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection. Eukaryot. Cell 2:10531060.
53. Sharkey, L. L.,, W. L. Liao,, A. K. Ghosh, and, W. A. Fonz. 2005. Flanking direct repeats of HisG alter URA3 marker expression at the HWP1 locus of Candida albicans. Microbiology 151:10611071.
54. Sia, R. A.,, K. B. Lengeler, and, J. Heitman. 2000. Diploid strains of the pathogenic basidiomycete Cryptococcus neoformans are thermally dimorphic. Fungal Genet. Biol. 29:153163.
55. Staab, J. F., and, P. Sundstrom. 2003. URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes. Trends Microbiol. 11:6973.
56. 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. 22:26682678.
57. 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.
58. Washburn, M. P.,, D. Wolters, and, J. R. Yates III. 2001. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19:242247.
59. Washburn, M. P.,, R. R. Ulaszek, and, J. R. Yates III. 2003. Reproducibility of quantitative proteomic analyses of complex biological mixtures by multidimensional protein identification technology. Anal. Chem. 75:50545061.
60. Wellington, M., and, E. Rustchenko. 2005. 5-Fluoro-orotic acid induces chromosome alterations in Candida albicans. Yeast 22:5770.
61. Wilson, R. B.,, D. Davis,, B. M. Enloe, and, A. P. Mitchell. 2000. A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions. Yeast 16:6570.
62. Winzeler, E. A.,, D. D. Shoemaker,, A. Astromoff,, H. Liang,, K. Anderson, et al. 1999. Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel analysis. Science 285:901906.
63. Wolter, D. A.,, M. P. Washburn, and, J. R. Yates. 2001. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73:56835690.
64. Wuchty, S. 2002. Interaction and domain networks of yeast. Proteomics 2:17151723.
65. Wuchty, S., and, E. Almaas. 2005. Peeling the yeast protein network. Proteomics 5:444449.
66. Yin, Z.,, D. Stead,, L. Selway,, J. Walker,, I. Riba-Garcia,, T. McLnerney,, S. Gaskell,, S. G. Oliver,, P. Cash, and, A. J. Brown. 2004. Proteomic response to amino acid starvation in Candida albicans and Saccharomyces cerevisiae. Proteomics 4:24252436.
67. Zhu, H.,, M. Bilgin,, R. Bangham,, D. Hall,, A. Casamayor,, P. Bertone,, N. Lan,, R. Jansen,, S. Bidlingmaier,, T. Houfek,, T. Mitchell,, P. Miller,, R. A. Dean,, M. Gerstein, and, M. Snyder. 2001. Global analysis of protein activities using proteome chips. Science 293:21012105.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error