RNA interference in parasitic nematodes: development of a toolkit for functional genomics

Nematode parasites are roundworms which cause severe disease in animals. As they infect many types of farm animals, they are responsible for significant economic losses in the United Kingdom and worldwide, particularly in sheep and cattle. At the moment, infection is controlled by treating the animals with drugs known as anthelmintics. The roundworms are quickly becoming resistant to these drugs, so we urgently need to develop new means of controlling infection, either by designing new drugs or developing vaccines. In order to do this we need to define new 'targets'; ie roundworm molecules which are crucial for their ability to infect, develop and survive inside animals. This type of research has been hindered by the lack of means to examine the function of roundworm molecules, which is normally perfomed by manipulating their genes. This proposal aims to develop a method of interfering with specific genes in a roundworm which can be easily manipulated in the laboratory. By the end of the project, we would hope to be in a position to apply these methods on a larger scale. This will greatly aid our understanding of the special features which allow these roundworms to act as parasites, and will be a powerful new tool in the development of new drugs and vaccines.

This application aims to establish standardised conditions for reliable post-transcriptional gene silencing in a parasitic nematode by RNA interference, and assay effects on parasite development in vivo. The species selected, Nippostrongylus brasiliensis, is ideal for this purpose as it is phylogenetically closely related to a range of parasites which cause serious disease in livestock, and is highly amenable to manipulation in the laboratory. The aim is thus to establish a model system for strongylid nematodes which is suitable for medium throughput analysis, so that it can be applied to a) defining the role of gene products in parasitism, and b) analysis of proteins of potential interest as drug and vaccine targets. We will develop standard conditions for effective RNAi in N. brasiliensis using a test panel of selected genes/mRNA targets which would be predicted to show phenotypic effects in vitro or in vivo upon silencing, or which are of interest from the perspectives of parasitism and vaccine deveopment. The target genes have different patterns of expression which will allow us to examine RNAi at different stages of parasite development. We will investigate electroporation with dsRNA (L1, activated L3 and adult stage parasites) and feeding (L1 to L3) with E. coli expressing dsRNA, or a combination of both. Prior to the first approach we will optimise conditions for electroporation of different stages of N. brasiliensis using firefly luciferase mRNA. An additional strategy will be to examine the effect of co-silencing orthologues of eri-1 and rrf-3, endogenous inhibitors of RNAi. The dynamics and longevity of silencing each target will be investigated in vitro prior to analysing the effect on parasite survival in vivo. For the latter, we will infect rats with parasites in which each gene has been silenced in turn, monitoring the progress of invasion and development, including parasite fecundity and the dynamics of expulsion from the intestinal tract.