RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs

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 Lleucine-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.

Excretion/secretion products from Schistosoma mansoni adults, eggs and schistosomula have unique peptidase specificity profiles

“…Adults also produce at least two metallo-peptidases, one of which is a metallo di-aminopeptidase activity, thus underscoring the utility of the MSP-MS approach to not only detect endopeptidase activity but also exopeptidase activity. Neither of the metallo-peptidase specificity profiles identified are similar to those recorded for a M17 family leucine aminopeptidase activity previously characterized in S. mansoni [84, 85]. …”

Purification and characterization of a Cys-Gly hydrolase from the gastropod mollusk,Patella caerulea

“…Recently, metallopeptidases belonging to the M17 family have been reported to be involved in parasitic infections in humans [37][38][39][40] ; they help parasites to survive by acting in the preliminary stages of their development, as, for example, eggs hatch in Schistosoma 41 or the maturation of immature trophozoites in P. falciparum 42 . Thus, the search for inhibitors of this enzyme activity has been proposed as a promising strategy for the treatment of various parasitic diseases.…”

Non-coding RNAs in schistosomes: an unexplored world

“…The RNAi approach was already used in several areas of S. mansoni biology (Boyle et al 2003, Skelly et al 2003, Correnti et al 2005, Delcroix et al 2006, Dinguirard and Yoshino 2006, Freitas et al 2007, Krautz-Peterson and Skelly 2008b, Morales et al 2008, Pereira et al 2008, Faghiri and Skelly 2009, Rinaldi et al 2009, Beckmann et al 2010 as well as in S. japonicum (Cheng et al 2005, Zhao et al 2008, Kumagai et al 2009. RNAi has proved itself as an important tool to elucidate gene function in schistosomes, in a similar way as in other organisms.…”