RNAi technology targeting PbGP43 and PbP27 in Paracoccidioides brasiliensis

Isaura Torres Gómez, Orville Hernandez Ruiz, Jose F. Muñoz, Ana María Garcia, Angela Restrepo, Juan G. McEwen

Unidad de Biología Celular y Molecular, Corporación para Investigaciones Biológicas (CIB), Medellín, Colombia.

DOI: 10.4236/ojgen.2013.32A2001                

Abstract

Efficient technologies for gene silencing would be important to carry out functional analysis with P. brasiliensis genes, as well as for a better understanding of the biology and pathogenesis of this pathogenic fungus. Due to the fact that homologous recombination is unusual in P. brasiliensis, the development of knockout isolates is currently non-feasible. The goal of this work was to assess RNA interference (RNAi) technology as an alternative tool for gene silencing previously employed successfully in H. capsulatum. For this purpose, we built different inverted repeat transgenic hairpin constructs to down-regulate the PbGP43 and PbP27 genes known to codify for two fungal immunogenic proteins that elicit a strong immune response during experimental paracoccidioidomycosis. Using the RNAi strategy, a reduction in the mRNA levels of the PbGP43 and PbP27 genes was observed during the first 20 days after selection; however, in the transformed yeast cells, the gene silencing status proved non-stable through the assay. We demonstrated that electrotransformation was suitable to transform P. brasiliensis yeast cells and integrate the hairpin constructions; nonetheless, gene silencing was not stable along the experimental time. A detailed analysis of the underlying molecular RNAi machinery may provide further insights into the intracellular mechanism that governs this reverse genetic tool.

Keywords

Paracoccidioides brasiliensis; Interference RNA; Gene Silencing; PbGP43; PbP27; Gene Expression

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature, 431, 343-349. doi:10.1038/nature02873
[2]	Appasani, K. (2005) RNA interference technology: From basic science to drug development. Cambridge University Press, New York, 1-13.
[3]	Sebghati, T.S., Engle, J.T. and Goldman, W.E. (2000) Intracellular parasitism by Histoplasmacapsulatum: Fungal virulence and calcium dependence. Science, 290, 1368-1372. doi:10.1126/science.290.5495.1368
[4]	Brandhorst, T.T., Wuthrich, M., Warner, T. and Klein, B. (1999) Targeted gene disruption reveals an adhesin indispensable for pathogenicity of Blastomycesdermatitidis. The Journal of Experimental Medicine, 189, 1207-1216. doi:10.1084/jem.189.8.1207
[5]	Brummer, E., Castaneda, E. and Restrepo, A. (1993) Paracoccidioidomycosis: An update. Clinical Microbiology Reviews, 6, 89-117.
[6]	Sturme, M.H., Puccia, R., Goldman, G.H. and Rodrigues, F. (2011) Molecular biology of the dimorphic fungi Paracoccidioides spp. Fungalbiol Reviews, 25, 89-97. doi:10.1016/j.fbr.2011.04.002
[7]	Puccia, R., Schenkman, S., Gorin, P.A. and Travassos, L.R. (1986) Exocellular components of Paracoccidioides brasiliensis: Identification of a specific antigen. Infection and Immunity, 53, 199-206.
[8]	McEwen, J.G., Ortiz, B.L., Garcia, A.M., Florez, A.M., Botero, S. and Restrepo, A. (1996) Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. Fungal Genetics and Biology, 20, 125-131. doi:10.1006/fgbi.1996.0027
[9]	De Camargo, Z., Unterkircher, C., Campoy, S.P. and Travassos, L.R. (1988) Production of Paracoccidioides brasiliensisexoantigens for immunodiffusion tests. Journal of Clinical Microbiology, 26, 2147-2151.
[10]	Camargo, Z.P., Berzaghi, R., Amaral, C.C. and Silva, S.H. (2003) Simplified method for producing Paracoccidioides brasiliensis exoantigens for use in immunodiffusion tests. Medical Mycology, 41, 539-542. doi:10.1080/13693780310001615358
[11]	Puccia, R. and Travassos, L.R. (1991) The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: Excretion and susceptibility to proteolysis. Archives of BiochemBiophys, 289, 298-302. doi:10.1016/0003-9861(91)90475-X
[12]	Garcia Blanco, S., Munoz, J.F., Torres, I., Diez Posada, S., Gomez, B.L., McEwen, J.G., Restrepo, S. and Garcia, A.M. (2011) Differential PbP27 expression in the yeast and mycelial forms of the Paracoccidioides brasiliensis species complex. Fungal Genetics and Biology, 48, 1087-1095. doi:10.1016/j.fgb.2011.09.001
[13]	Sambrook, J. and Russell, D.W. (2001) Molecular cloning: A laboratory manual. Harbor Laboratory Press, New York, Cold Spring.
[14]	Galagan, J.E., Calvo, S.E., Borkovich, K.A., Selker, E.U., Read, N.D., Jaffe, D., FitzHugh, W., Ma, L.J., Smirnov, S., Purcell, S., Rehman, B., Elkins, T., Engels, R., Wang, S., Nielsen, C.B., Butler, J., Endrizzi, M., Qui, D., Ianakiev, P., Bell-Pedersen, D., Nelson, M.A., WernerWashburne, M., Selitrennikoff, C.P., Kinsey, J.A., Braun, E.L., Zelter, A., Schulte, U., Kothe, G.O., Jedd, G., Mewes, W., Staben, C., Marcotte, E., Greenberg, D., Roy, A., Foley, K., Naylor, J., Stange-Thomann, N., Barrett, R., Gnerre, S., Kamal, M., Kamvysselis, M., Mauceli, E., Bielke, C., Rudd, S., Frishman, D., Krystofova, S., Rasmussen, C., Metzenberg, R.L., Perkins, D.D., Kroken, S., Cogoni, C., Macino, G., Catcheside, D., Li, W., Pratt, R.J., Osmani, S.A., DeSouza, C.P., Glass, L., Orbach, M.J., Berglund, J.A., Voelker, R., Yarden, O., Plamann, M., Seiler, S., Dunlap, J., Radford, A., Aramayo, R., Natvig, D.O., Alex, L.A., Mannhaupt, G., Ebbole, D.J., Freitag, M., Paulsen, I., Sachs, M.S., Lander, E.S., Nusbaum, C. and Birren, B. (2003) The genome sequence of the filamentous fungus Neurosporacrassa. Nature, 422, 859-868. doi:10.1038/nature01554
[15]	Finn, R.D., Clements, J. and Eddy, S.R. (2011) HMMER web server: Interactive sequence similarity searching. Nucleic Acids Research, 39, W29-W37. doi:10.1093/nar/gkr367
[16]	Woods, J.P. and Goldman, W.E. (1993) Autonomous replication of foreign DNA in Histoplasmacapsulatum: Role of native telomeric sequences. Journal of Bacteriology, 175, 636-641.
[17]	Rappleye, C.A., Engle, J.T., and Goldman W.E. (2004) RNA interference in Histoplasmacapsulatum demonstrates a role for alpha-(1,3)-glucan in virulence. Molecular Microbiology, 53, 153-165. doi:10.1111/j.1365-2958.2004.04131.x
[18]	Zhang, Y., Li, G., He, D., Yu, B., Yokoyama, K. and Wang L. (2011) Efficient insertional mutagenesis system for the dimorphic pathogenic fungus Sporothrixschenckiiusing Agrobacterium tumefaciens. Journal of Microbiological Methods, 84, 418-422. doi:10.1016/j.mimet.2011.01.017
[19]	vanBurik, J.A., Schreckhise, R.W., White, T.C., Bowden, R.A. and Myerson D. (1998) Comparison of six extraction techniques for isolation of DNA from filamentous fungi. Medical Mycology, 36, 299-303. doi:10.1080/02681219880000471
[20]	Goldman, G.H., dos Reis Marques, E., Duarte Ribeiro, D.C., de Souza Bernardes, L.A., Quiapin, A.C., Vitorelli, P.M., Savoldi, M., Semighini, C.P., de Oliveira, R.C., Nunes, L.R., Travassos, L.R., Puccia, R., Batista, W.L., Ferreira, L.E., Moreira, J.C., Bogossian, A.P., Tekaia, F., Nobrega, M.P., Nobrega, F.G. and Goldman M.H. (2003) Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: Identification of putative homologues of Candida albicans virulence and pathogenicity genes. Eukaryotical Cell, 2, 34-48. doi:10.1128/EC.2.1.34-48.2003
[21]	Livak, K.J. and Schmittgen T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-408. doi:10.1006/meth.2001.1262
[22]	Desjardins, C.A., Champion, M.D., Holder, J.W., Muszewska, A., Goldberg, J., Bailao, A.M., Brigido, M.M., Ferreira, M.E., Garcia, A.M., Grynberg, M., Gujja, S., Heiman, D.I., Henn, M.R., Kodira, C.D., Leon-Narvaez, H., Longo, L.V., Ma, L.J., Malavazi, I., Matsuo, A.L., Morais, F.V., Pereira, M., Rodriguez-Brito, S., Sakthikumar, S., Salem-Izacc, S.M., Sykes, S.M., Teixeira, M.M., Vallejo, M.C., Walter, M.E., Yandava, C., Young, S., Zeng, Q., Zucker, J., Felipe, M.S., Goldman, G.H., Haas, B.J., McEwen, J.G., Nino-Vega, G., Puccia, R., San-Blas, G., Soares, C.M., Birren, B.W. and Cuomo C.A. (2011) Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis. PLoS Genet, 7, e1002345. doi:10.1371/journal.pgen.1002345
[23]	Almeida, A.J., Cunha, C., Carmona, J.A., SampaioMarques, B., Carvalho, A., Malavazi, I., Steensma, H.Y., Johnson, D.I., Leao, C., Logarinho, E., Goldman, G.H., Castro, A.G., Ludovico, P. and Rodrigues, F. (2009) Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis. Fungal Genetics and Biology, 46, 919-926. doi:10.1016/j.fgb.2009.08.004
[24]	Hernandez, O., Almeida, A.J., Gonzalez, A., Garcia, A.M., Tamayo, D., Cano, L.E. Restrepo, A. and McEwen, J.G. (2010) A 32-kilodalton hydrolase plays an important role in Paracoccidioides brasiliensis adherence to host cells and influences pathogenicity. Infection and Immunity, 78, 5280-5286. doi:10.1128/IAI.00692-10
[25]	Ruiz, O.H., Gonzalez, A., Almeida, A.J., Tamayo, D., Garcia, A.M., Restrepo, A. and McEwen, J.G. (2011) Alternative oxidasemediatespathogenresistance in Paracoccidioides brasiliensis infection. PLOS Neglected Tropical Diseases, 5, e1353. doi:10.1371/journal.pntd.0001353
[26]	Tamayo, D., Munoz, J.F., Torres, I., Almeida, A.J., Restrepo, A., McEwen, J.G. and Hernandez, O. (2013) Involvement of the 90 kDaheatshockproteinduring adaptation of Paracoccidioides brasiliensis to different environmental conditions. Fungal Genetics and Biology, 51, 34-41. doi:10.1016/j.fgb.2012.11.005