Impact of Paracoccin Gene Silencing on Paracoccidioides brasiliensis Virulence

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Fernandes, Fabricio F. ; Oliveira, Aline F. ; Landgraf, Taise N. ; Cunha, Cristina ; Carvalho, Agostinho ; Vendruscolo, Patricia E. ; Goncales, Relber A. ; Almeida, Fausto ; da Silva, Thiago A. ; Rodrigues, Fernando ; Roque-Barreira, Maria Cristina

• Document type: Journal article
• Source: MBIO; v. 8, n. 4 JUL-AUG 2017.
• Web of Science Citations: 5

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. (AU)

• FAPESP's process: 16/00629-4 - Characterization of enzymatic and lectin domains and the effects on innate immunity cell receptors
• Grantee: Fabrício Freitas Fernandes
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/05359-0 - Distribution of paracoccin on the surface of Paracoccidioides brasiliensis and interaction with innate immunity cell receptors: role in the acute phase of paracoccidioidomycosis
• Grantee: Aline Ferreira de Oliveira Pereira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/22561-7 - Overexpression of paracoccin: phenotype and virulence of Paracoccidioides brasiliensis strains
• Grantee: Relber Aguiar Gonçales
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 16/04877-2 - Design of new therapeutic strategies, based on the carbohydrate recognition, against cryptococcosis
• Grantee: Thiago Aparecido da Silva
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 12/09611-0 - Effect of lectin ArtinM on murine CD4+ T and CD8+ T cells
• Grantee: Thiago Aparecido da Silva
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 13/04088-0 - Lectin from pathogens
• Grantee: Maria Cristina Roque Antunes Barreira
• Support Opportunities: Research Projects - Thematic Grants