TITLE:
RNAi technology targeting PbGP43 and PbP27 in Paracoccidioides brasiliensis
AUTHORS:
Isaura Torres Gómez, Orville Hernandez Ruiz, Jose F. Muñoz, Ana María Garcia, Angela Restrepo, Juan G. McEwen
KEYWORDS:
Paracoccidioides brasiliensis; Interference RNA; Gene Silencing; PbGP43; PbP27; Gene Expression
JOURNAL NAME:
Open Journal of Genetics,
Vol.3 No.2B,
July
22,
2013
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.