Direct experimental manipulation of intestinal cells in Ascaris suum , with minor influences on the global transcriptome

Abstract

Ascaris suum provides a powerful model for studying parasitic nematodes, including individual tissues such as the intestine, an established target for anthelmintic treatments. Here, we add a valuable experimental component to our existing functional, proteomic, transcriptomic and phylogenomic studies of the Ascaris suum intestine, by developing a method to manipulate intestinal cell functions via direct delivery of experimental treatments (in this case, double-stranded (ds)RNA) to the apical intestinal membrane. We developed an intestinal perfusion method for direct, controlled delivery of dsRNA/heterogeneous small interfering (hsi) RNA into the intestinal lumen for experimentation. RNA-Seq (22 samples) was used to assess influences of the method on global intestinal gene expression. Successful mRNA-specific knockdown in intestinal cells of adult A. suum was accomplished with this new experimental method. Global transcriptional profiling confirmed that targeted transcripts were knocked down more significantly than any others, with only 12 (0.07% of all genes) or 238 (1.3%) off-target gene transcripts consistently differentially regulated by dsRNA treatment or the perfusion experimental design, respectively (after 24 h). The system supports controlled, effective delivery of treatments (dsRNA/hsiRNA) to the apical intestinal membrane with relatively minor off-target effects, and builds on our experimental model to dissect A. suum intestinal cell functions with broad relevance to parasitic nematodes.

Omics Driven Understanding of the Intestines of Parasitic Nematodes

“…The ability of the intestinal perfusion method to support experimental manipulation of intestinal cell functions has been tested using dsRNA as the treatment for several A. suum intestinal genes (Rosa et al, 2017). The genes used in this study were selected based on a number of criteria, including evidence of results with dsRNA from orthologous genes in heterologous species.…”

“…It is important to evaluate off-target dsRNA effects by RNAseq and challenges they might present for interpretation of experimental results even when effectiveness of dsRNA delivery and knockdown of intestinal cell transcripts is established. The A. suum dsRNA perfusion study (Rosa et al, 2017) quantified global transcript effects for treatments of dsRNA constructs from two different genes, then control decapitated worms at time 0 or cannulated for 24 h, and anterior and posterior regions of worms from each treatment group. By comparison to other species/model systems, only modest effects were observed for up- or downregulation of off-target genes related to dsRNA treatments.…”

“…By comparison to other species/model systems, only modest effects were observed for up- or downregulation of off-target genes related to dsRNA treatments. For instance, transcripts for only eight and four intestinal genes showed significant up- or downregulation, respectively, by both dsRNA gene constructs in both anterior and posterior intestinal regions (Rosa et al, 2017). Additional, but still modest, effects were observed related to individual dsRNA gene treatments and then anterior and posterior intestinal regions.…”

Genomics of the Parasitic Nematode Ascaris and Its Relatives

“…Analysis of the intestinal transcriptome shows many unique and highly expressed transcripts are present in the anterior region [ 45 ]. Together with proteomics [ 92 ] and the development of Ascaris intestinal cannulation and a perfusion model [ 93 ], an experimental system was established for testing and validation of drug candidates. The Ascaris intestine model represents an important system for developing novel anthelmintic drugs against nematode intestinal cells [ 91 , 94 ].…”

Nematode Small RNA Pathways in the Absence of piRNAs

“…Ascaris primary siRNAs. Several studies suggest that introduction of dsRNA leads to RNA interference in Ascaris embryos (Davis, R.E., unpublished), larvae [102,103], the isolated and perfused adult intestine [104], and muscle [105] through injection of dsRNA into the pseudocoelom of adults. The key C. elegans RDE-1 Argonaute binds exogenous siRNAs derived from dsRNA.…”