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 S. 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 SmLAP1 and SmLAP2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP2 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 >or=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.

Figure 1

A schematic view of the genome loci encoding the Schistosoma mansoni leucine aminopeptidase (LAP) genes. The genome browser view, obtained from www.genedb.org/schistosoma/, indicates the position of the exons of SmLAP1 and SmLAP2 (coding portions in light blue boxes, 3′ untranslated regions in white boxes). High scoring regions on tblastx comparisons against the S. japonicum genome draft are indicated (grey boxes). Stage specific assembled ESTs (color coded) mapped with the PASA package [60] on the genomic sequence and their orientations are indicated.

Figure 2

Alignment of amino acid sequences of leucine aminopeptidases. Exon alternation in genomic sequences is indicated by alternation of blue and black type. Conserved regions are shown as shaded blocks. Residues that participate in coordination of metal ions are indicated in red and those involved in the catalysis are arrowed. (By way of reference, residues Asp 289, Asp 367 and Glu 369 [SmLAP1 numbering] are involved in the coordination of a zinc atom, while Asp 289, Lys 284, Asp 307 and Glu 369 bind a second zinc ion. Lys 296 and Arg 371 are also involved in catalysis.) The conserved carboxy terminal domain is indicated by underlining in the alignment positions. The reference sequences are from Fasciola hepatica (accession Q17TZ3) Caenorhabditis elegans (Q27245) and Homo sapiens (Q6P0L6). The schistosome identifiers correspond to those provided in the genome assembly 4 of Schistosoma mansoni and assembly 2 of Schistosoma japonicum. The two contigs comprising SjLAP1 are included.

Figure 3

Phylogenetic tree reveals relationships between schistosome leucine aminopeptidases and other family M17 LAP enzymes. Neighbor joining tree based on the conserved carboxy terminal domain of selected proteins. The aminopeptidase A from Escherichia coli served as the outgroup. Bootstrap values supporting the major clusters are indicated. Branch names indicate species or common name along with Uniprot accessions.

Figure 4

Developmental stage specific expression of leucine aminopeptidases (LAP) in Schistosoma mansoni. Panel A. cDNAs from eggs (E), miracidia (M), sporocysts (Sp), cercariae (C), schistosomules (So), mix sex adults (A), male adults (♂) and female adults (♀) were employed as templates for PCRs using primers specific for the SmLAP1, SmLAP2 and S. mansoni actin genes. Panel B. Densitometric measurements (arbitrary units) across the different bands and relative levels of SmLAP1 (blue) and SmLAP2 (red) compared with levels for actin presented in panel A.

Figure 5

Knockdown of egg hatching by RNA interference in Schistosoma mansoni. Hatching of eggs was blocked by dsRNA against SmLAP1, SmLAP2, both together, and bestatin. A. Representative micrographs of eggs during the hatching process, one hour after incubation in water. A: mock control, B: dsLUC treated eggs, C: dsLAP1 treated eggs, D: dsLAP2 treated eggs, E: eggs treated with dsLAP1 and dsLAP2, F: bestatin treated eggs. Bar, 200 μm. B. Percentage of hatched eggs: 100%, control eggs. Statistically significant differences were evident among the dsLAP1 group, dsLAP2 group, dsLAP1+dsLAP2 group, bestatin group and the controls mock and irrelevant dsRNA. The bars indicate standard deviation of mean for two experiments, and asterisks indicate significant differences (P<0.01).

Figure 6

RNAi targeting schistosome leucine aminopeptidases results in statistically significant knock down of protease activities and mRNA levels. Panel A. Leucine aminopeptidase (LAP) activity assay performed using the diagnostic substrate, L-leucine-7-amido-4-methylcoumarin hydroxide. LAP activity in relative fluorescence units per microgram (RLU/μg) of soluble schistosome protein presented on Y-axis. Groups of eggs were treated with double stranded RNA targeting SmLAP1, SmLAP2, both, firefly luciferase, and not treated with dsRNA (mock control). Panel B. Semi-quantitative RT-PCR using LAP specific primers and actin as an internal control. Schistosome egg cDNAs were synthesized from serial dilutions of total RNAs (100 ng, 10 ng, 1 ng) isolated from treatment group of eggs, soaked in doubled strand RNA targeting SmLAP1, SmLAP2, both, firefly luciferase, and not treated with dsRNA (mock control). The bars show the standard deviations for two experiments. Asterisks indicate significant differences (P<0.01) between treated groups and both controls groups. The difference between the mock control and the irrelevant dsRNA control also was significant (P<0.01).