New Developments of RNAi in Paracoccidioides brasiliensis: Prospects for High-Throughput, Genome-Wide, Functional Genomics

Abstract

BackgroundThe Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis.Methodology/Principal FindingsIn this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach.Conclusions/SignificanceWe have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.

Characterization of the Paracoccidioides Hypoxia Response Reveals New Insights into Pathogenesis Mechanisms of This Important Human Pathogenic Fungus

“…There are a reduced number of works relating the functional analysis of genes in Paracoccidioides in the last six years because the achievement of viable and stable mutants of Paracoccidioides spp. is a hard task [ 11 , 13 , 89 – 94 ]. This parsimony in functional analysis surely reflects the complexity of those studies in the genus Paracoccidioides , as well as in other pathogenic fungi.…”

New insights into a complex fungal pathogen: the case of Paracoccidioides spp.

“…In a later attempt to improve RNAi technology in Paracoccidioides , successful knockdown of GP43 was achieved through the expression of intron‐containing hairpin RNA (ihpRNA). Employing this strategy reduced Gp43 transcript levels by 73.1% (Goes et al ., ).…”

Liver damage in schistosomiasis is reduced by adipose tissue-derived stem cell therapy after praziquantel treatment

“…However, the effect of stem cells on the inflammatory response associated with parasitic diseases has not been well investigated yet. Recently, the effect of stem cells on parasitic diseases such as malaria [35] and Chagas [36][37][38][39][40][41] was reported. However, only a few preview studies have analyzed the effect of MSC on Schistosoma infection [42][43][44][45][46].…”