1. RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs
- Author
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Yousef N. Alrefaei, Paul J. Brindley, Gabriel Rinaldi, John P. Dalton, José F. Tort, Estela Castillo, Martín Cancela, and Maria E. Morales
- Subjects
Molecular Sequence Data ,Article ,Schistosoma japonicum ,Leucyl Aminopeptidase ,RNA interference ,Gene Duplication ,parasitic diseases ,Animals ,Amino Acid Sequence ,Molecular Biology ,Leucyl aminopeptidase ,Gene ,Phylogeny ,Gene knockdown ,Exopeptidase activity ,Genes, Essential ,Sequence Homology, Amino Acid ,biology ,Intron ,Helminth Proteins ,Schistosoma mansoni ,biology.organism_classification ,Molecular biology ,Cell biology ,Gene Knockdown Techniques ,RNA Interference ,Parasitology - Abstract
Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the Schistosoma mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP 1 and 2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP 2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate L-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by ≥ 80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases.
- Published
- 2009