Kinetic analysis of gene expression during mycelium to yeast transition and yeast to mycelium germination in Paracoccidioides brasiliensis
Keywords:
Paracoccidioides, paracoccidioidomicosis, levaduras, micelio, germinación, expresión génica
Abstract
Introduction: Paracoccidioidomycosis is an endemic systemic mycosis caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus that in tissues and cultures at 37 °C grows as a yeast while at lower temperatures (less than 24 °C) it becomes a mold; however the genes that rule these processes and their expression are poorly understood.
Objective: This research focused on the kinetic expression of certain genes in P. brasiliensis throughout the dimorphic process, one that involves the transition from the mycelium to yeast forms and the germination from the yeast to mycelium form.
Materials and methods: A real-time quantitative polymerase chain reaction (RT-qPCR) was optimized to measure the expression of ten genes connected with diverse cellular functions including cell synthesis and wall structure, oxidative stress response, heat shock response, metabolism, proteins' processing, solute transport across the cell membrane and signal transduction pathways at different time points during the mycelia to yeast transition, as well as in the yeast to mycelia germination processes.
Results: Genes involved in cell synthesis and wall structure, metabolism and signal transduction were differentially expressed and highly up-regulated during the yeast to mycelia germination process; on the other hand, genes involved in heat shock response, cell synthesis and wall structure were highly up-regulated during the mycelia to yeast transition process. The remaining genes were differentially regulated during both processes.
Conclusion: In this work the up-regulation of certain genes involved in the morphological changes occurring in P. brasiliensis yeast and mycelia forms were confirmed, indicating that these biological
processes play an important role during the host-pathogen interactions, as well as in the fungus adaptation to environmental conditions.
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References
1. Restrepo A, McEwen JG, Castaneda E. The habitat of Paracoccidioides brasiliensis: how far from solving the riddle? Med Mycol. 2001;39:233-41.
2. Restrepo A, Tobón A. Paracoccidioides brasiliensis. In: Mandell B, Bennett J, Dolin R, editors. Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases. 7th ed. Philadelphia: Churchill Livingston, Elsevier; 2009. p. 3357-64.
3. Klein BS, Tebbets B. Dimorphism and virulence in fungi. Curr Opin Microbiol. 2007;10:314-9.
4. Restrepo A, Tobón A, Agudelo C. Paracoccidioidomycosis. In: Hospenthal A, Rinaldi M, editors. Infectious Diseases: diagnosis and treatment of human mycoses. Totowa, New Jersey: Humana Press; 2008. p. 331- 42.
5. Brummer E, Castaneda E, Restrepo A. Paracoccidioidomycosis: an update. Clin Microbiol Rev. 1993;6:89-117.
6. Loose D, Stover E, Restrepo A, Stevens D, Feldman D. Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis. Proc Natl Acad Sci USA. 1983;80:7659-63.
7. Stover E, Schar G, Clemons K, Stevens D, Feldman D. Estradiol-binding proteins from mycelial and yeast-form cultures of Paracoccidioides brasiliensis. Infect Immun. 1986;51:199-203.
8. McEwen J, Bedoya V, Patino M, Salazar M, Restrepo A. Experimental murine paracoccidiodomycosis induced by the inhalation of conidia. J Med Vet Mycol. 1987;25:165-75.
9. Bagagli E, Theodoro RC, Bosco SM, McEwen JG. Paracoccidioides brasiliensis: phylogenetic and ecological aspects. Mycopathologia. 2008;165:197-207.
10. Casadevall A, Steenbergen JN, Nosanchuk JD. ‘Ready made' virulence and ‘dual use' virulence factors in pathogenic environmental fungi--the Cryptococcus neoformans paradigm. Curr Opin Microbiol. 2003; 6:332-7.
11. Bastos KP, Bailao AM, Borges CL, Faria FP, Felipe MS, Silva MG, et al. The transcriptome analysis of early morphogenesis in Paracoccidioides brasiliensis mycelium reveals novel and induced genes potentially associated to the dimorphic process. BMC microbiol. 2007;7:29.
12. Nunes LR, Costa de Oliveira R, Leite DB, da Silva VS, dos Reis Marques E, da Silva Ferreira ME, et al. Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition. Eukaryot Cell. 2005;4:2115-28.
13. Goldman GH, dos Reis Marques E, Duarte Ribeiro DC, de Souza Bernardes LA, Quiapin AC, Vitorelli PM, et al. Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence and pathogenicity genes. Eukaryot Cell. 2003;2:34-48.
14. Garcia AM, Hernandez O, Aristizabal BH, De Souza Bernardes LA, Puccia R, Naranjo TW, et al. Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell. Med Mycol. 2010;48:147-54.
15. Queiroz-Telles F. Paracoccidioides brasiliensis: Ultrastructural findings. In: Franco M, Restrepo A, Del Negro G, editors. Paracoccidioidomycosis. Boca Raton, FL.: CRC Press; 1994.p. 27-48.
16. Ramirez Martinez J. Paracoccidioides brasiliensis: conversion of yeastlike forms into mycelia in submerged culture. J Bacteriol. 1971;105:523-6.
17. Restrepo BI, McEwen JG, Salazar ME, Restrepo A. Morphological development of the conidia produced by Paracoccidioides brasiliensis mycelial form. J Med Vet Mycol. 1986;24:337-9.
18. Garcia AM, Hernandez O, Aristizabal BH, Cano LE, Restrepo A, McEwen JG. Identification of genes associated with germination of conidia to form mycelia in the fungus Paracoccidioides brasiliensis. Biomedica. 2009;29:403-12.
19. Torres FA, Vilaca R, Pepe De Moraes LM, Reis VC, Felipe MS. Expression of a kexin-like gene from the human pathogenic fungus Paracoccidioides brasiliensis in Saccharomyces cerevisiae. Med Mycol. 2008;46:385-8.
20. Venancio EJ, Daher BS, Andrade RV, Soares CM, Pereira IS, Felipe MS. The kex2 gene from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis. Yeast. 2002;19:1221-31.
21. Venancio EJ, Kyaw CM, Mello CV, Silva SP, Soares CM, Felipe MS, et al. Identification of differentially expressed transcripts in the human pathogenic fungus Paracoccidioides brasiliensis by differential display. Med Mycol. 2002;40:45-51.
22. Enderlin CS, Selitrennikoff CP. Cloning and characterization of a Neurospora crassa gene required for (1,3) beta-glucan synthase activity and cell wall formation. Proc Natl Acad Sci USA. 1994;91:9500-4.
23. Rappleye CA, Eissenberg LG, Goldman WE. Histoplasma capsulatum alpha-(1,3)-glucan blocks innate immune recognition by the beta-glucan receptor. Proc Natl Acad Sci USA. 2007;104:1366-70.
24. Rappleye CA, Engle JT, Goldman WE. RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence. Mol Microbiol. 2004;53:153-65.
25. Sorais F, Barreto L, Leal JA, Bernabe M, San-Blas G, Nino-Vega GA. Cell wall glucan synthases and GTPases in Paracoccidioides brasiliensis. Med Mycol. 2010;48:35-47.
26. Van Aken O, Giraud E, Clifton R, Whelan J. Alternative oxidase: a target and regulator of stress responses. Physiol Plant. 2009;137:354-61.
27. Restrepo A, Jimenez B. Growth of Paracoccidioides brasiliensis yeast phase in a chemically defined culture medium. J Clin Microbiol. 1980;12:279-81.
28. Calich VL, Kipsnis TL, Mariano M, Fava Neto C, Da Silva D. The activation of the complement system by Paracoccidioidis brasiliensis in vitro: Its opsonic efect and possible significance for an in vivo model of infection. Clin Immunol Immunopathol. 1979;12:20-30.
29. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-8.
30. Jakob U, Buchner J. Assisting spontaneity: the role of Hsp90 and small Hsps as molecular chaperones. Trends Biochem Sci. 1994;19:205-11.
31. Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet. 1988;22:631-77.
32. Nadeau K, Das A, Walsh CT. Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases. The Journal of Biological Chemistry. 1993;268:1479-87.
33. Felipe MS, Andrade RV, Petrofeza SS, Maranhao AQ, Torres FA, Albuquerque P, et al. Transcriptome haracterization of the dimorphic and pathogenic fungus aracoccidioides brasiliensis by EST analysis. Yeast. 003;20:263-71.
34. Nicola AM, Andrade RV, Dantas AS, Andrade PA,Arraes FB, Fernandes L, et al. The stress responsive and morphologically regulated hsp90 gene from Paracoccidioides brasiliensis is essential to cell viability. BMC Microbiol. 2008;8:158.
35. Shapiro RS, Uppuluri P, Zaas AK, Collins C, Senn H, Perfect JR, et al. Hsp90 orchestrates temperaturedependent Candida albicans morphogenesis via Ras1-PKA signaling. Curr Biol. 2009;19:621-9.
36. Parente JA, Borges CL, Bailao AM, Felipe MS, Pereira M, de Almeida Soares CM. Comparison of transcription of multiple genes during mycelia transition to yeast cells of Paracoccidioides brasiliensis reveals insights to fungal differentiation and pathogenesis. Mycopathologia. 2008;165:259-73.
37. Gessler NN, Aver'yanov AA, Belozerskaya TA. Reactive oxygen species in regulation of fungal development. Biochemistry (Mosc). 2007;72:1091-109.
38. Saijo T, Miyazaki T, Izumikawa K, Mihara T, Takazono T, Kosai K, et al. Skn7p is involved in oxidative stress response and virulence of Candida glabrata. Mycopathologia. 2010;169:81-90.
39. He XJ, Mulford KE, Fassler JS. Oxidative stress function of the Saccharomyces cerevisiae Skn7 receiver domain. Eukaryot Cell. 2009;8:768-78.
40. Stock AM, Robinson VL, Goudreau PN. Two-component signal transduction. Annu Rev Biochem. 2000;69:183-215.
41. Sinclair DA, Dawes IW. Genetics of the synthesis of serine from glycine and the utilization of glycine as sole nitrogen source by Saccharomyces cerevisiae. Genetics. 1995;140:1213-22.
42. Pao GM, Saier MH Jr. Response regulators of bacterial signal transduction systems: selective domain shuffling during evolution. J Mol Evol. 1995;40:136-54.
43. Walmsley AR, Barrett MP, Bringaud F, Gould GW. Sugar transporters from bacteria, parasites and mammals: structure-activity relationships. Trends Biochem Sci. 1998;23:476-81.
44. Steiner HY, Naider F, Becker JM. The PTR family: a new group of peptide transporters. Mol Microbiol. 1995;16:825-34.
45. Seidah NG, Chretien M. Eukaryotic protein processing: endoproteolysis of precursor proteins. Curr Opin Biotechnol. 1997;8:602-7.
46. Oka T, Hamaguchi T, Sameshima Y, Goto M, Furukawa K. Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans. Microbiology. 2004;150:1973-82.
47. Almeida AJ, Carmona JA, Cunha C, Carvalho A, Rappleye CA, Goldman WE, et al. Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis. Fungal Genet Biol. 2007;44:1387-98.
2. Restrepo A, Tobón A. Paracoccidioides brasiliensis. In: Mandell B, Bennett J, Dolin R, editors. Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases. 7th ed. Philadelphia: Churchill Livingston, Elsevier; 2009. p. 3357-64.
3. Klein BS, Tebbets B. Dimorphism and virulence in fungi. Curr Opin Microbiol. 2007;10:314-9.
4. Restrepo A, Tobón A, Agudelo C. Paracoccidioidomycosis. In: Hospenthal A, Rinaldi M, editors. Infectious Diseases: diagnosis and treatment of human mycoses. Totowa, New Jersey: Humana Press; 2008. p. 331- 42.
5. Brummer E, Castaneda E, Restrepo A. Paracoccidioidomycosis: an update. Clin Microbiol Rev. 1993;6:89-117.
6. Loose D, Stover E, Restrepo A, Stevens D, Feldman D. Estradiol binds to a receptor-like cytosol binding protein and initiates a biological response in Paracoccidioides brasiliensis. Proc Natl Acad Sci USA. 1983;80:7659-63.
7. Stover E, Schar G, Clemons K, Stevens D, Feldman D. Estradiol-binding proteins from mycelial and yeast-form cultures of Paracoccidioides brasiliensis. Infect Immun. 1986;51:199-203.
8. McEwen J, Bedoya V, Patino M, Salazar M, Restrepo A. Experimental murine paracoccidiodomycosis induced by the inhalation of conidia. J Med Vet Mycol. 1987;25:165-75.
9. Bagagli E, Theodoro RC, Bosco SM, McEwen JG. Paracoccidioides brasiliensis: phylogenetic and ecological aspects. Mycopathologia. 2008;165:197-207.
10. Casadevall A, Steenbergen JN, Nosanchuk JD. ‘Ready made' virulence and ‘dual use' virulence factors in pathogenic environmental fungi--the Cryptococcus neoformans paradigm. Curr Opin Microbiol. 2003; 6:332-7.
11. Bastos KP, Bailao AM, Borges CL, Faria FP, Felipe MS, Silva MG, et al. The transcriptome analysis of early morphogenesis in Paracoccidioides brasiliensis mycelium reveals novel and induced genes potentially associated to the dimorphic process. BMC microbiol. 2007;7:29.
12. Nunes LR, Costa de Oliveira R, Leite DB, da Silva VS, dos Reis Marques E, da Silva Ferreira ME, et al. Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition. Eukaryot Cell. 2005;4:2115-28.
13. Goldman GH, dos Reis Marques E, Duarte Ribeiro DC, de Souza Bernardes LA, Quiapin AC, Vitorelli PM, et al. Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence and pathogenicity genes. Eukaryot Cell. 2003;2:34-48.
14. Garcia AM, Hernandez O, Aristizabal BH, De Souza Bernardes LA, Puccia R, Naranjo TW, et al. Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell. Med Mycol. 2010;48:147-54.
15. Queiroz-Telles F. Paracoccidioides brasiliensis: Ultrastructural findings. In: Franco M, Restrepo A, Del Negro G, editors. Paracoccidioidomycosis. Boca Raton, FL.: CRC Press; 1994.p. 27-48.
16. Ramirez Martinez J. Paracoccidioides brasiliensis: conversion of yeastlike forms into mycelia in submerged culture. J Bacteriol. 1971;105:523-6.
17. Restrepo BI, McEwen JG, Salazar ME, Restrepo A. Morphological development of the conidia produced by Paracoccidioides brasiliensis mycelial form. J Med Vet Mycol. 1986;24:337-9.
18. Garcia AM, Hernandez O, Aristizabal BH, Cano LE, Restrepo A, McEwen JG. Identification of genes associated with germination of conidia to form mycelia in the fungus Paracoccidioides brasiliensis. Biomedica. 2009;29:403-12.
19. Torres FA, Vilaca R, Pepe De Moraes LM, Reis VC, Felipe MS. Expression of a kexin-like gene from the human pathogenic fungus Paracoccidioides brasiliensis in Saccharomyces cerevisiae. Med Mycol. 2008;46:385-8.
20. Venancio EJ, Daher BS, Andrade RV, Soares CM, Pereira IS, Felipe MS. The kex2 gene from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis. Yeast. 2002;19:1221-31.
21. Venancio EJ, Kyaw CM, Mello CV, Silva SP, Soares CM, Felipe MS, et al. Identification of differentially expressed transcripts in the human pathogenic fungus Paracoccidioides brasiliensis by differential display. Med Mycol. 2002;40:45-51.
22. Enderlin CS, Selitrennikoff CP. Cloning and characterization of a Neurospora crassa gene required for (1,3) beta-glucan synthase activity and cell wall formation. Proc Natl Acad Sci USA. 1994;91:9500-4.
23. Rappleye CA, Eissenberg LG, Goldman WE. Histoplasma capsulatum alpha-(1,3)-glucan blocks innate immune recognition by the beta-glucan receptor. Proc Natl Acad Sci USA. 2007;104:1366-70.
24. Rappleye CA, Engle JT, Goldman WE. RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence. Mol Microbiol. 2004;53:153-65.
25. Sorais F, Barreto L, Leal JA, Bernabe M, San-Blas G, Nino-Vega GA. Cell wall glucan synthases and GTPases in Paracoccidioides brasiliensis. Med Mycol. 2010;48:35-47.
26. Van Aken O, Giraud E, Clifton R, Whelan J. Alternative oxidase: a target and regulator of stress responses. Physiol Plant. 2009;137:354-61.
27. Restrepo A, Jimenez B. Growth of Paracoccidioides brasiliensis yeast phase in a chemically defined culture medium. J Clin Microbiol. 1980;12:279-81.
28. Calich VL, Kipsnis TL, Mariano M, Fava Neto C, Da Silva D. The activation of the complement system by Paracoccidioidis brasiliensis in vitro: Its opsonic efect and possible significance for an in vivo model of infection. Clin Immunol Immunopathol. 1979;12:20-30.
29. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-8.
30. Jakob U, Buchner J. Assisting spontaneity: the role of Hsp90 and small Hsps as molecular chaperones. Trends Biochem Sci. 1994;19:205-11.
31. Lindquist S, Craig EA. The heat-shock proteins. Annu Rev Genet. 1988;22:631-77.
32. Nadeau K, Das A, Walsh CT. Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases. The Journal of Biological Chemistry. 1993;268:1479-87.
33. Felipe MS, Andrade RV, Petrofeza SS, Maranhao AQ, Torres FA, Albuquerque P, et al. Transcriptome haracterization of the dimorphic and pathogenic fungus aracoccidioides brasiliensis by EST analysis. Yeast. 003;20:263-71.
34. Nicola AM, Andrade RV, Dantas AS, Andrade PA,Arraes FB, Fernandes L, et al. The stress responsive and morphologically regulated hsp90 gene from Paracoccidioides brasiliensis is essential to cell viability. BMC Microbiol. 2008;8:158.
35. Shapiro RS, Uppuluri P, Zaas AK, Collins C, Senn H, Perfect JR, et al. Hsp90 orchestrates temperaturedependent Candida albicans morphogenesis via Ras1-PKA signaling. Curr Biol. 2009;19:621-9.
36. Parente JA, Borges CL, Bailao AM, Felipe MS, Pereira M, de Almeida Soares CM. Comparison of transcription of multiple genes during mycelia transition to yeast cells of Paracoccidioides brasiliensis reveals insights to fungal differentiation and pathogenesis. Mycopathologia. 2008;165:259-73.
37. Gessler NN, Aver'yanov AA, Belozerskaya TA. Reactive oxygen species in regulation of fungal development. Biochemistry (Mosc). 2007;72:1091-109.
38. Saijo T, Miyazaki T, Izumikawa K, Mihara T, Takazono T, Kosai K, et al. Skn7p is involved in oxidative stress response and virulence of Candida glabrata. Mycopathologia. 2010;169:81-90.
39. He XJ, Mulford KE, Fassler JS. Oxidative stress function of the Saccharomyces cerevisiae Skn7 receiver domain. Eukaryot Cell. 2009;8:768-78.
40. Stock AM, Robinson VL, Goudreau PN. Two-component signal transduction. Annu Rev Biochem. 2000;69:183-215.
41. Sinclair DA, Dawes IW. Genetics of the synthesis of serine from glycine and the utilization of glycine as sole nitrogen source by Saccharomyces cerevisiae. Genetics. 1995;140:1213-22.
42. Pao GM, Saier MH Jr. Response regulators of bacterial signal transduction systems: selective domain shuffling during evolution. J Mol Evol. 1995;40:136-54.
43. Walmsley AR, Barrett MP, Bringaud F, Gould GW. Sugar transporters from bacteria, parasites and mammals: structure-activity relationships. Trends Biochem Sci. 1998;23:476-81.
44. Steiner HY, Naider F, Becker JM. The PTR family: a new group of peptide transporters. Mol Microbiol. 1995;16:825-34.
45. Seidah NG, Chretien M. Eukaryotic protein processing: endoproteolysis of precursor proteins. Curr Opin Biotechnol. 1997;8:602-7.
46. Oka T, Hamaguchi T, Sameshima Y, Goto M, Furukawa K. Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans. Microbiology. 2004;150:1973-82.
47. Almeida AJ, Carmona JA, Cunha C, Carvalho A, Rappleye CA, Goldman WE, et al. Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis. Fungal Genet Biol. 2007;44:1387-98.
How to Cite
1.
Hernández O, Tamayo D, Torres I, Restrepo Ángela, McEwen JG, García AM. Kinetic analysis of gene expression during mycelium to yeast transition and yeast to mycelium germination in Paracoccidioides brasiliensis. biomedica [Internet]. 2011 Jun. 30 [cited 2024 May 16];31(4):570-9. Available from: https://revistabiomedica.org/index.php/biomedica/article/view/441
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