Impact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence

ABSTRACT Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, se...

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Autores principales:	Fabrício F. Fernandes, Aline F. Oliveira, Taise N. Landgraf, Cristina Cunha, Agostinho Carvalho, Patrícia E. Vendruscolo, Relber A. Gonçales, Fausto Almeida, Thiago A. da Silva, Fernando Rodrigues, Maria Cristina Roque-Barreira
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	ATMT fungal virulence Paracoccidioides brasiliensis paracoccidioidomycosis paracoccin paracoccin silencing Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/6e460be652b0426299f673f1e642b671
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spelling	oai:doaj.org-article:6e460be652b0426299f673f1e642b6712021-11-15T15:51:44ZImpact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence10.1128/mBio.00537-172150-7511https://doaj.org/article/6e460be652b0426299f673f1e642b6712017-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00537-17https://doaj.org/toc/2150-7511ABSTRACT Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, several recent studies have employed the methodology of gene modulation in P. brasiliensis using antisense RNA (AsRNA) and Agrobacterium tumefaciens-mediated transformation (ATMT) to identify proteins that influence fungus virulence. Our previous observations suggested that paracoccin (PCN), a multidomain fungal protein with both lectin and enzymatic activities, may be a potential P. brasiliensis virulence factor. To explore this, we used AsRNA and ATMT methodology to obtain three independent PCN-silenced P. brasiliensis yeast strains (AsPCN1, AsPCN2, and AsPCN3) and characterized them with regard to P. brasiliensis biology and pathogenicity. AsPCN1, AsPCN2, and AsPCN3 showed relative PCN expression levels that were 60%, 40%, and 60% of that of the wild-type (WT) strain, respectively. PCN silencing led to the aggregation of fungal cells, blocked the morphological yeast-to-mycelium transition, and rendered the yeast less resistant to macrophage fungicidal activity. In addition, mice infected with AsPCN1, AsPCN2, and AsPCN3 showed a reduction in fungal burden of approximately 96% compared with those inoculated with the WT strain, which displayed a more extensive destruction of lung tissue. Finally, mice infected with the PCN-silenced yeast strains had lower mortality than those infected with the WT strain. These data demonstrate that PCN acts as a P. brasiliensis contributory virulence factor directly affecting fungal pathogenesis. IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis, the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens-mediated transformation system, provides new strategies for studying P. brasiliensis. Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis, we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis.Fabrício F. FernandesAline F. OliveiraTaise N. LandgrafCristina CunhaAgostinho CarvalhoPatrícia E. VendruscoloRelber A. GonçalesFausto AlmeidaThiago A. da SilvaFernando RodriguesMaria Cristina Roque-BarreiraAmerican Society for MicrobiologyarticleATMTfungal virulenceParacoccidioides brasiliensisparacoccidioidomycosisparacoccinparacoccin silencingMicrobiologyQR1-502ENmBio, Vol 8, Iss 4 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	ATMT fungal virulence Paracoccidioides brasiliensis paracoccidioidomycosis paracoccin paracoccin silencing Microbiology QR1-502
spellingShingle	ATMT fungal virulence Paracoccidioides brasiliensis paracoccidioidomycosis paracoccin paracoccin silencing Microbiology QR1-502 Fabrício F. Fernandes Aline F. Oliveira Taise N. Landgraf Cristina Cunha Agostinho Carvalho Patrícia E. Vendruscolo Relber A. Gonçales Fausto Almeida Thiago A. da Silva Fernando Rodrigues Maria Cristina Roque-Barreira Impact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence
description	ABSTRACT Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, several recent studies have employed the methodology of gene modulation in P. brasiliensis using antisense RNA (AsRNA) and Agrobacterium tumefaciens-mediated transformation (ATMT) to identify proteins that influence fungus virulence. Our previous observations suggested that paracoccin (PCN), a multidomain fungal protein with both lectin and enzymatic activities, may be a potential P. brasiliensis virulence factor. To explore this, we used AsRNA and ATMT methodology to obtain three independent PCN-silenced P. brasiliensis yeast strains (AsPCN1, AsPCN2, and AsPCN3) and characterized them with regard to P. brasiliensis biology and pathogenicity. AsPCN1, AsPCN2, and AsPCN3 showed relative PCN expression levels that were 60%, 40%, and 60% of that of the wild-type (WT) strain, respectively. PCN silencing led to the aggregation of fungal cells, blocked the morphological yeast-to-mycelium transition, and rendered the yeast less resistant to macrophage fungicidal activity. In addition, mice infected with AsPCN1, AsPCN2, and AsPCN3 showed a reduction in fungal burden of approximately 96% compared with those inoculated with the WT strain, which displayed a more extensive destruction of lung tissue. Finally, mice infected with the PCN-silenced yeast strains had lower mortality than those infected with the WT strain. These data demonstrate that PCN acts as a P. brasiliensis contributory virulence factor directly affecting fungal pathogenesis. IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis, the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens-mediated transformation system, provides new strategies for studying P. brasiliensis. Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis, we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis.
format	article
author	Fabrício F. Fernandes Aline F. Oliveira Taise N. Landgraf Cristina Cunha Agostinho Carvalho Patrícia E. Vendruscolo Relber A. Gonçales Fausto Almeida Thiago A. da Silva Fernando Rodrigues Maria Cristina Roque-Barreira
author_facet	Fabrício F. Fernandes Aline F. Oliveira Taise N. Landgraf Cristina Cunha Agostinho Carvalho Patrícia E. Vendruscolo Relber A. Gonçales Fausto Almeida Thiago A. da Silva Fernando Rodrigues Maria Cristina Roque-Barreira
author_sort	Fabrício F. Fernandes
title	Impact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence
title_short	Impact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence
title_full	Impact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence
title_fullStr	Impact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence
title_full_unstemmed	Impact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence
title_sort	impact of paracoccin gene silencing on <italic toggle="yes">paracoccidioides brasiliensis</italic> virulence
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/6e460be652b0426299f673f1e642b671
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Impact of Paracoccin Gene Silencing on <italic toggle="yes">Paracoccidioides brasiliensis</italic> Virulence

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