Impact of Paracoccin Gene Silencing on
Paracoccidioides brasiliensis
Virulence
Authors: Fernandes Fabrício F. , Oliveira Aline F. , Landgraf Taise N. , Cunha Cristina , Carvalho Agostinho , Vendruscolo Patrícia E. , Gonçales Relber A. , Almeida Fausto , da Silva Thiago A. , Rodrigues Fernando , Roque-Barreira Maria Cristina 
Published: July 2017

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 (As
PCN1
, As
PCN2
, and As
PCN3
) and characterized them with regard to
P. brasiliensis
biology and pathogenicity. As
PCN1
, As
PCN2
, and As
PCN3
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 As
PCN1
, As
PCN2
, and As
PCN3
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.