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

<div><p>Background</p><p>The Fungal Genome Initiative of the Broad Institute, in partnership with the <i>Paracoccidioides</i> 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 <i>P. brasiliensis</i>.</p><p>Methodology/Principal Findings</p><p>In this paper, we report <i>Agrobacterium tumefaciens</i> 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 <i>Streptoalloteichus hindustanus</i> bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in <i>P. brasiliensis</i>. 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.</p><p>Conclusions/Significance</p><p>We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future <i>Paracoccidioides</i> spp. functional genomics research.</p></div

Cassettes assembled in the present work.

<p>(<b>A</b>). Cassette for the expression of the ZeoR marker under the transcriptional regulation of Prm_CBP1 and Ttr_GP43,which was used in preliminary ATMT of <i>P. brasiliensis</i>. (<b>B–D</b>). Cassettes assembled to validate Prm_Act and Prm_GP43against Prm_CBP1 through the expression of the bifunctional selective marker “Shble::mCherry” in yeast cells. (<b>E</b>). Purpose-built cAlfG, adapted for the Gateway Technology, intended for the expression of intron_GP43-containing hairpin RNA to trigger RNAi in yeast cells. Abbreviations: Ttr, transcription termination region of <i>Pb18</i> GP43 gene; Prm_GP43, promoter region of <i>Pb18</i> GP43 gene; Prm_Act, promoter region of <i>Pb18</i> actin gene; Prm_CBP1, promoter region of <i>H. capsulatum</i> CBP1 gene; Rfc, gateway reading frame cassette; Intr, first intron of the <i>Pb18</i> GP43 gene.</p

Epifluorescence microscopy analysis of yeast cells expressing the bifunctional selective marker.

<p>Yeast cells carrying integrated cassettes under the transcriptional regulation of Prm_CBP1 (<b>A</b>), Prm_Act (<b>B</b>) or Prm_GP43 (<b>C</b>) were collected from late-log phase cultures and visualized through confocal microscopy. (D). Yeast cells carrying the integrated “Prm_CBP1::<i>Shble</i>::Ttr_GP43” expression cassette was used as negative control. Scale bar: 10 µm.</p

RNA interference of <i>Pb18</i> GP43 gene expression triggered artificially with intron-containing dsRNA.

<p>(<b>A</b>). GP43 gene expression levels were investigated in six cAlf-GP43 transformants in relation to cAlfG-control by qRT-PCR after 8 months of subculturing in non-selective media. Results were normalized against the internal control L34. (<b>B–C</b>) Epifluorescence microscopy analysis of transformant C4 (C), in which the GP43 gene expression was nearly abolished, in comparison to cAlfG-control (<b>B</b>). Scale bar: 10 µm.</p

ATMT of <i>P. brasiliensis</i> to certify the Zeocin/ZeoR positive selection system and the terminal GP43 gene transcription region.

<p>(<b>A</b>). Four independent transformation experiments were performed by co-cultivating 1×10⁶ viable <i>Pb18</i> yeast cells with <i>A. tumefaciens</i> at different ratios. <i>A. tumefaciens</i> carried the “Prm_CBP1::<i>Shble</i>::Ttr_GP43” cassette to express the ZeoR selection marker (*, P = 0.0002; **, P = 0.001). (<b>B</b>). Three independent transformation experiments were performed by co-cultivating 1×10⁶ viable <i>Pb18</i> yeast cells with <i>A. tumefaciens</i> at a ratio of 1∶100 and recovering the yeast cells using two different selective media (*, P = 0.02).</p

Proliferation and viability of hASCs cultured on TCPS and electrospun PHB/PHB-HV fiber mesh.

<p>(A) MTT proliferation assays performed 7, 14 and 21 days after the cell seeding and cultured in two specific medium: the basal medium and the endothelial differentiation medium. The results are expressed as the means ± SD, (*) indicating a significant difference with p<0.05, (**) p<0,01, (***) p<0,001; (B) cell viability after 21 days of cell culture with the basal medium and (C) the endothelial differentiation medium on the electrospun PHB/PHB-HV fiber mesh, as analyzed by Calcein-AM staining.</p

<i>A. tumefaciens</i> mediated transformation efficiency of <i>Pb18</i> yeast cells with cassettes under the transcriptional regulation of different promoters.

<p>(<b>A</b>). Three independent ATMT experiments were performed to verify if the transformation efficiency of <i>Pb18</i> yeast cells could be further improved by expressing the bifunctional selective marker under the transcriptional regulation of Prm_Act or Prm_GP43 in relation to Prm_CBP1. Data analysis showed non-significant differences (CBP1 vs. Actin, P = 0.17; CBP1 vs. GP43, P = 0.55). (<b>B</b>). The gMFI of the bifunctional selective marker “Shble::mCherry”, which was expressed under the transcriptional regulation of Prm_CBP1, Prm_Act or Prm_GP43, was determined for yeast cell populations in late log-phase growth. Control refers to yeast cell populations transformed with the cassette “Prm_CBP1::<i>Shble</i>::Ttr_GP43”. The gMFI values were normalized in respect to control yeast cell populations expressing only the ZeoR selection marker. (*, P = 0.01).</p

RT-PCR analysis of VEGFR2 mRNA expression during endothelial differentiation on the electrospun PHB/PHB-HV fiber mesh and TCPS.

<p>Total RNA was extracted from cells cultured on basal medium and endothelial differentiation medium for analysis of VEGFR2 mRNA expression. The cells were cultivated up to 21 days.</p

Flow cytometry analysis of hASCs.

<p>The expression pattern of specific antigens on the surface of the hASCs is depicted with representative histograms and the expression of each marker. The cell population expressed CD29, CD44, CD73 and HLA-ABC, and did not express CD34, CD45 and HLA-DR.</p

Protein expression of hASCs during endothelial differentiation.

<p>Confocal images of the expression of the VE-Cadherin (A) and the vWF factor (B) after 21 days. A.1 and B.1 - hASCs cultured with the basal medium on the TCPS coverslips, A.2 and B.2 - hASCs cultured with the endothelial differentiation medium on the TCPS coverslips, A.3 and B.3 - hASCs cultured with the basal medium on the electrospun PHB/PHB-HV fiber mesh, A.4 and B.4 - hASCs cultured with the endothelial differentiation medium on the electrospun PHB/PHB-HV fiber mesh. Scale bar 20 µm. The images, A.2, A.4, B.2 and B.4 represent the overlay of bright-field and confocal images for visualization of the fiber mesh. (C) Fluorescence intensity of the expression of VE-Cadherin and the vWF factor in cells differentiated on TCPS coverslip and electrospun PHB/PHB-HV fiber mesh. The results are expressed as the means ± SD, (*) significant difference for p<0,05. (a.u): arbitrary units.</p