The Transcriptome of Schistosoma mansoni Developing Eggs Reveals Key Mediators in Pathogenesis and Life Cycle Propagation

Abstract

Schistosomiasis, the most important helminthic disease of humanity, is caused by infection with parasitic flatworms of the genus Schistosoma. The disease is driven by parasite eggs becoming trapped in host tissues, followed by inflammation and granuloma formation. Despite abundant transcriptome data for most developmental stages of the three main human-infective schistosome species-Schistosoma mansoni, S. japonicum and S. haematobium-the transcriptomic profiles of developing eggs remain under unexplored. In this study, we performed RNAseq of S. mansoni eggs laid in vitro during early and late embryogenesis, days 1-3 and 3-6 post-oviposition, respectively. Analysis of the transcriptomes identified hundreds of up-regulated genes during the later stage, including venom allergen-like (VAL) proteins, well-established host immunomodulators, and genes involved in organogenesis of the miracidium larva. In addition, the transcriptomes of the in vitro laid eggs were compared with existing publicly available RNA-seq datasets from S. mansoni eggs collected from the livers of rodent hosts. Analysis of enriched GO terms and pathway annotations revealed cell division and protein synthesis processes associated with early embryogenesis, whereas cellular metabolic processes, microtubule-based movement, and microtubule cytoskeleton organization were enriched in the later developmental time point. This is the first transcriptomic analysis of S. mansoni embryonic development, and will facilitate our understanding of infection pathogenesis, miracidial development and life cycle progression of schistosomes.

Keywords: RNAseq; Schistosoma mansoni; early embryogenesis; eggs; late embryogenesis; neglected tropical disease (NTD).

Figure 1

(A) Timeline depicting the experimental design. On day 0 (D0) adult worms perfused from infected mice were washed and placed in culture for three days. The worms laid eggs, i.e. in vitro laid eggs (IVLE), for three days (bracket). On day 3 (D3) the worms were removed from the culture, and half of the IVLE were collected for RNAseq - D3, early embryogenesis sample. The remaining IVLE were cultured for three more days, and on day 6 (D6) they were collected for RNAseq - D6, late embryogenesis sample. (B, C) Representative micrographs of 3 days old- (B) and 6 days old- (C) in vitro laid eggs (IVLE). Scale bar: 100 μm. em, embryo (yellow arrow); yk, yolk (red arrow); white arrowhead, fully developed egg containing the mature miracidium. (D) Clustering of egg samples using Principal Component Analysis. D3, D3 IVLE; D6, D6 IVLE; Li, liver eggs (E) Clustering of egg samples using Sample Distance Matrix. Names of samples are described in Supplementary Table S1.

Figure 2. Differential gene expression among egg samples.

(A) Hierarchical clustering showing divergent transcriptomic signatures among the three samples. Liver, liver eggs; D3, D3 IVLE; D6, D6 IVLE. The colour scale indicates the Z-score values. (B) Volcano plots showing differentially expressed genes (DEGs) in D3 IVLE compared to liver eggs (left) and in D6 IVLE compared to D3 IVLE (right). Highlighted genes: Saposin (Smp_105420), kappa-5 (left: Smp_335470, Smp_335480 & Smp_335490; right: Smp_335490 & Smp_344300); Omega-1 (left: Smp_334170 & Smp_345790; right: Smp_345790), Hsp20 (Smp_302270), VAL (venom-allergen like protein - Smp_070250, Smp_176180 & Smp_120670), a-GAL (Alpha-N-acetylgalactosaminidase - Smp_247750 & Smp_247760), Cathepsin B (Smp_067060 & Smp_103610). (C) Volcano plot showing DEGs in D6 IVLE compared to liver eggs. Highlighted genes: kappa-5 (Smp_335470 & Smp_335480), MEA (major egg antigen - Smp_302350), Omega-1 (Smp_334170), TUBA1A (Tubulin alpha-1A chain - Smp_090120). In the volcano plots the x-axes represent -log10Padj values and the y-axes represent log2FoldChange. The volcano plots are available as interactive charts at http://schisto.xyz/IVLE/.

Figure 3

Heatmaps showing the relative gene expression of the egg antigens IPSE, Omega-1, kappa-5 and VALs in liver, D3 and D6 IVLE, as indicated. The colour scale shows relative values to the mean of each row using normalised counts from DESeq2.

Figure 4

Gene Ontology (GO) enrichment in differentially expressed genes. For each comparison - D3 vs Liver; D6 vs D3; D6 vs Liver, only GO terms with FDR<0.01 and at least three genes are visualised. D3, D3 IVLE; D6, D^{^} IVLE; Liver, liver eggs.