Developmental gene expression profiles of the human pathogen Schistosoma japonicum

Abstract

Background: The schistosome blood flukes are complex trematodes and cause a chronic parasitic disease of significant public health importance worldwide, schistosomiasis. Their life cycle is characterised by distinct parasitic and free-living phases involving mammalian and snail hosts and freshwater. Microarray analysis was used to profile developmental gene expression in the Asian species, Schistosoma japonicum. Total RNAs were isolated from the three distinct environmental phases of the lifecycle -- aquatic/snail (eggs, miracidia, sporocysts, cercariae), juvenile (lung schistosomula and paired but pre-egg laying adults) and adult (paired, mature males and egg-producing females, both examined separately). Advanced analyses including ANOVA, principal component analysis, and hierarchal clustering provided a global synopsis of gene expression relationships among the different developmental stages of the schistosome parasite.

Results: Gene expression profiles were linked to the major environmental settings through which the developmental stages of the fluke have to adapt during the course of its life cycle. Gene ontologies of the differentially expressed genes revealed a wide range of functions and processes. In addition, stage-specific, differentially expressed genes were identified that were involved in numerous biological pathways and functions including calcium signalling, sphingolipid metabolism and parasite defence.

Conclusion: The findings provide a comprehensive database of gene expression in an important human pathogen, including transcriptional changes in genes involved in evasion of the host immune response, nutrient acquisition, energy production, calcium signalling, sphingolipid metabolism, egg production and tegumental function during development. This resource should help facilitate the identification and prioritization of new anti-schistosome drug and vaccine targets for the control of schistosomiasis.

Figure 1

The complex lifecycle of Schistosoma japonicum involves distinct free-living and parasitic stages (see text for details). The numbers indicate the seven developmental stages investigated by microarray and real time PCR analysis.

Figure 2

Hierarchal clustering by gene and developmental stage. Four clusters of up-regulated genes were identified: 1, Aquatic/snail stages; 2 and 3, Adult female worms; 4. Adult male worms. Gene expression is shown in the heat map as up-regulated (Red) down-regulated (Green) or no change (Black). E, eggs; M, miracidia; S, sporocysts; C, cercariae; L, lung schistosomula; F4, juvenile females; M4, juvenile males; F6, F6.5, F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge; M6, M6.5, M7, adult male worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge.

Figure 3

Gene Ontology (GO) analysis showing the correlation between GO categories and microarray expression data calculated using ErmineJ software. The GO annotations with parent-child analysis are presented on the left. Contributions to each of the categories from each lifecycle stage (2-fold or higher gene expression) are shaded. The overall number of genes in each category represented on the microarray and the correlation p-value associated with the entire lifecycle are shown on the right. NA, the parent category is significant but a child category is not. Genes expressed in adult parasites from weeks 6–7.

Figure 4

Validation of selected transcripts in different developmental stages of S. japonicum. The real time PCR analysis is presented as bar graphs and is shown in copy number for each gene and stage. The corresponding microarray gene expression data are presented below the bar graphs as heat maps, with up-regulated genes shown in red, down-regulated genes shown in green and unchanged genes shown in black. E, eggs; M, miracidia; S, sporocysts; C,: cercariae; L, lung schistosomula; F4, juvenile females; M4, juvenile males; F6, F6.5, F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge; M6, M6.5, M7, adult male worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge.

Figure 5

Principal component analysis of the ANOVA showing an overview of differentially expressed genes in the different developmental stages of S. japonicum analysed. E, eggs; M, miracidia; S, sporocysts; C,: cercariae; L, lung schistosomula; F4, juvenile females; M4, juvenile males; F6, F6.5, F7, adult female worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge; M6, M6.5, M7, adult male worms analysed at 6, 6.5 and 7 weeks post-cercarial challenge.