Impact of Paracoccin Gene Silencing on Paracoccidioides brasiliensis 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, 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.

Paracoccin overexpression in Paracoccidioides brasiliensis reveals the influence of chitin hydrolysis on fungal virulence and host immune response

“…Concerning the mice survival to the fungal infection, we observed that all animals infected with wt-PCN yeasts survived throughout a 40 days post-infection period, whereas the ones infected with ov-PCN yeasts started dying at day 27 post-infection and none survived to a 35 days post-infection period (Fig 3F). We concluded that PCN overexpression aggravates the disease caused by P. brasiliensis yeasts, reinforcing the notion that PCN acts as a P. brasiliensis virulence factor [18].…”

“…Recently, we have shown that PCN-silenced yeasts of P. brasiliensis are unable to perform the morphological transition from yeast to mycelium [18]. Herein, we studied the transition of ov-PCN yeasts by quantitative optical microscopy, for 7 days (168 h) following the shift in culture temperature from 37 to 25 °C.…”

“…Because the P. brasiliensis PCN-knocked down yeasts are less virulent than the wt-PCN yeasts [18], we hypothesized that overexpression of PCN could promote different pathogenic profile of yeast in mice. The lungs of BALB/c mice, inoculated through the intranasal route with 2×10 6 cells of ov-PCN or wt-PCN yeasts, were microscopically examined, thirty days after infection, regarding the extension of the granulomatous lesions.…”

“…are poorly known owing to the lack of adequate characterization of fungal components. Among them, we highlight the glycoprotein gp43 [6-10], Hsp60 [11-13], Pb40 [14], Pb27 [15], SconCp [16], Cdc42p [17] and paracoccin (PCN) [18-20]. We described PCN from P. brasiliensis as a major protein component of a GlcNAc-binding fraction of yeast extracts [19, 20].…”

“…Recently, our group successfully knocked-down PCN expression. Working with the obtained P. brasiliensis transformed yeasts, we verified that PCN is essential for the fungal morphological transition and virulence [18]. To date, overexpression of endogenous components has not been explored in Paracoccidioides spp.…”