Final Activity Report Summary - COMBINATORIAL RNAI (The study of gene families in C. elegans by combinatioral RNA interference)
Over the past decade Caenorhabditis elegans has become a popular model organism for parasitic nematode research and many examples have been published to illustrate its use. It has especially been useful for the research on mechanisms of action of anthelmintics and on anthelmintic resistance. However, its validity in parasite vaccine research is less clear. It has been suggested that studying genes in C. elegans by RNA interference might help to identify new vaccine targets.
In addition, C. elegans might also be an interesting alternative expression system for nematode vaccine antigens. The objective of this project was to asses both these hypothesis. First, combinatorial RNAi was used to silence the C. elegans homologues of well-known intestinal vaccine candidates from the blood feeding parasites Haemonchus contortus and Ancylostoma caninum. These included the metallopeptidase III and pepsin of the H-gal-GP complex, the H11 aminopeptidase and the cysteine proteinases. L3/L4 stage worms were soaked in a mixture of dsRNA targeting these genes.
The RNAi silencing of the different genes was confirmed by RT-PCRs. Worms were visually examined over a period of 5 days for phenotypic effects such as growth, development, movement, feeding and reproduction. Surprisingly, silencing all these genes did not induce any visible phenotypes in the worms. The lack of phenotype might be explained by the fact that the genes targeted in this study are all members of multigene families and it is possible that genes within such families have some functional redundancy. Alternatively, the target genes were identified based on their sequence homology and although they are the closest homologues, there is no guarantee that their function is conserved with the parasite genes.
The data clearly infers that this screening methods would not identify gut expressed proteins as vaccine candidates against H. contortus. This, in turn, suggests the need for considerable caution in interpreting C. elegans-based data to identify potential parasite vaccine targets.
The second objective was to assess C. elegans as an expression for helminths antigens. As an example we have cloned the H. contortus cathepsin L protein. Recombinant Hc-CPL-1 with a polyhistidine tag added to the C-terminal was expressed in an active and glycosylated form in C. elegans. Optimal expression was obtained expressing Hc-cpl-1 under control of the promoter of the homologous C. elegans cpl-1 gene. The recombinant protein was purified from liquid cultures by nickel chelation chromatography in sufficient amounts for vaccination studies to be carried out.
This study provides proof of principle that active, post-translationally modified parasitic nematode proteases can be expressed in C. elegans and this approach can now be extended for expression of known protective antigens.
In addition, C. elegans might also be an interesting alternative expression system for nematode vaccine antigens. The objective of this project was to asses both these hypothesis. First, combinatorial RNAi was used to silence the C. elegans homologues of well-known intestinal vaccine candidates from the blood feeding parasites Haemonchus contortus and Ancylostoma caninum. These included the metallopeptidase III and pepsin of the H-gal-GP complex, the H11 aminopeptidase and the cysteine proteinases. L3/L4 stage worms were soaked in a mixture of dsRNA targeting these genes.
The RNAi silencing of the different genes was confirmed by RT-PCRs. Worms were visually examined over a period of 5 days for phenotypic effects such as growth, development, movement, feeding and reproduction. Surprisingly, silencing all these genes did not induce any visible phenotypes in the worms. The lack of phenotype might be explained by the fact that the genes targeted in this study are all members of multigene families and it is possible that genes within such families have some functional redundancy. Alternatively, the target genes were identified based on their sequence homology and although they are the closest homologues, there is no guarantee that their function is conserved with the parasite genes.
The data clearly infers that this screening methods would not identify gut expressed proteins as vaccine candidates against H. contortus. This, in turn, suggests the need for considerable caution in interpreting C. elegans-based data to identify potential parasite vaccine targets.
The second objective was to assess C. elegans as an expression for helminths antigens. As an example we have cloned the H. contortus cathepsin L protein. Recombinant Hc-CPL-1 with a polyhistidine tag added to the C-terminal was expressed in an active and glycosylated form in C. elegans. Optimal expression was obtained expressing Hc-cpl-1 under control of the promoter of the homologous C. elegans cpl-1 gene. The recombinant protein was purified from liquid cultures by nickel chelation chromatography in sufficient amounts for vaccination studies to be carried out.
This study provides proof of principle that active, post-translationally modified parasitic nematode proteases can be expressed in C. elegans and this approach can now be extended for expression of known protective antigens.