RNA Interference in Schistosoma mansoni Schistosomula: Selectivity, Sensitivity and Operation for Larger-Scale Screening

Abstract

BackgroundThe possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi.Methodology/Principal FindingsWe investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose- dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite.ConclusionsTransient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens.

Vaccinomics for the Major Blood Feeding Helminths of Humans

“…Parasitic helminths, by virtue of their often complex life cycles and large genomes, have rendered themselves refractory to many genetic manipulation tools that allow the exploration of gene function (Mann et al, , 2010. RNAi is now widely used to assess gene function in schistosomes, and appears to be particularly effective for genes expressed in tissues readily accessible to dsRNA, such as the tegument and gastrodermis (Krautz-Peterson et al, 2010;Stefanic et al, 2010). RNAi has been used to confirm gene function for a number of potential schistosome vaccine antigens and drug targets, and helps explain how some vaccines, which are based on these proteins, might exert their efficacy.…”

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

“…Whereas b 2 -integrins have been implicated in the recognition and binding of molecules originating from bacterial and yeast pathogens, viruses appear to use mainly b 3 -integrins (and to a lesser extent b 1 , b 5 or b 7 -integrins) for cell entry. [71] Intriguingly, Ac-NIF appears to specifically bind to the  subunit of complement receptor 3 [72] and localise to the I-domain of  M [73] that contains the metal ion-dependent adhesion site (MIDAS). [74] In terms of structural elucidation, the molecular interactions between viruses or viral proteins with integrins have been investigated in many instances, and include three-dimensional structures by electron microscopy or crystallography.…”

“…Both larval and adult worms of schistosomes can be maintained in culture in vitro and are amenable to RNA interference (RNAi). [72] In contrast to a number of other parasites, where RNAi has not been reported or inconsistent susceptibilities have been observed, [73] schistosomes, particularly S. mansoni, should allow integrated studies of SAPLIPs, combining functional explorations with elucidation of these proteins at the molecular, structural and/or immuno-biochemical levels. Conclusions regarding homologous structure-function relationships, from one individual SAPLIP to another, are not advisable for the reasons discussed in this review.…”

RNA Interference and Nonspecific Controls in Parasitic Helminths

“…In order to build up information to elucidate helminths biology and host-parasite interaction towards discovery of new drug targets, comes along the feasibility of systems and tools to manipulate gene expression to make sense of so much data generated by genome and transcriptome sequencing. Among very few techniques available for gene characterization in parasitic helminths, RNA interference (RNAi) is the most accessible and employed tool [3][4][5].…”

“…One obstacle of using this technique in helminths parasites has been the lack of consistency of knockdown, not only in different species, but also within genes in the same species [3][4][5]14]. Even though there have been several suggestions to explain those inconsistencies [15][16][17], there remain a number of studies concerning questions in which RNAi has not been successful and therefore, unpublished.…”