Characterization of the Schistosoma transcriptome opens up the world of helminth genomics
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Among the metazoan parasites that cause debilitating disease in man, schistosomes are the first group for which near-complete transcriptome complements have been described. This new genomic information will have an enormous impact on all future investigations into the biology, pathogenesis and control of schistosomiasis.
KeywordsSchistosomiasis Schistosoma Praziquantel Liver Fluke Schistosoma Japonicum
Schistosomes: the past and present
Schistosomes are members of the class Trematoda, a large, diverse and medically important group of parasitic worms within the phylum Platyhelminthes (flat worms). Ancestral trematodes were probably parasites of ancient marine molluscs. With the evolution of predatory vertebrate animals, however, the digenic forms of primitive trematodes developed complex life-cycles involving two or more different host species and both sexual and asexual reproduction. These parasites successfully diversified and expanded, accompanying the molluscs as they evolved into terrestrial and freshwater forms, and consequently all classes of vertebrate are now host to many trematode species. It is estimated that well in excess of 7,000 trematode species are parasitic on teleost fish, amphibians, reptiles, birds and mammals . Trematodes of medical or veterinary importance include Paragonimus westermani (human lung fluke), Clonorchis sinensis (human liver fluke), Fasciola hepatica (cattle liver fluke) and, most importantly, the schistosomes (blood flukes).
We know that schistosomiasis (or bilharzia) has been a scourge of man for tens of thousands of years. Schistosome eggs have been found in ancient mummies in both Egypt and China. In 1851, working in Egypt, Theodore Bilharz first reported the presence of adult schistosome worms in human blood vessels. Several individual species that are infective to man were subsequently described, including Schistosoma japonicum in South East Asia, by Katsurada in 1904, and S. mansoni and S. haematobium in Africa and the near East, by Sambon in 1907. These three species, of the five that cause human schistosomiasis, are responsible for the vast majority of human infections. Currently, it is estimated that at least 200 million human infections exist in 70 tropical and subtropical countries, leading to chronic debilitating disease and some 200-500 thousand human deaths per year . Although an effective anti-schistosome drug, praziquantel, has been available for 25 years, the number of human infections worldwide stubbornly refuses to decline. This is partly because treatment is short-term efficacious, such that people remain susceptible to reinfection. Worryingly, praziquantel is the only drug widely available for treatment of human schistosomiasis; its exact mode of action is unknown, and the possible development of parasite drug-resistance is a concern.
Schistosomes are beautifully adapted to each of the many diverse environments encountered during their life-cycle, including the human host. As a result, they are especially difficult parasites to control. For example, not only are S. mansoni worm pairs able to produce hundreds of eggs daily in the immunologically hostile blood environment, they also provoke host immune responses that are essential for the passage of their eggs from the blood, through the host gut wall, to the environment outside. Thus, schistosomes modulate the human immune system to survive in the blood for years and subvert it to facilitate egg migration to continue their life-cycle. The combination of this complex, dioecious life-cycle and the inaccessible site of the worms within the mammalian host has prevented the use of classical genetics to investigate the role of individual genes in the highly sophisticated relationships between schistosomes and their intermediate and definitive hosts.
Comparative transcriptome features of Schistosoma expressed sequence tags
The publication of the near-complete EST complements for S. japonicum  and S. mansoni  has added some 168,347 new schistosome sequences to GenBank . The submitted sequences were derived from cDNA libraries created from diverse parasite source materials using different construction techniques. The S. japonicum project  used a directional phage vector for cDNA-library construction and generated ESTs from two different parasite life-stages (adult worm and egg). In contrast, the S. mansoni project  used both a normalized adult worm cDNA library and ORF expressed sequence tag (ORESTES) [7, 8] mini-libraries from six life-cycle stages (adult, egg, miracidium, germ ball, cercaria, and cultured day-7 schistosomulum). Thus, stage-specific transcripts were more likely to be identified by the S. mansoni project, although a correlation cannot always be inferred between the number of EST reads for a given transcript in a specific life-cycle stage and differential expression .
The S. mansoni report  estimates, using two different methodologies, a total gene complement of between 13,960 and 14,205 unique elements. From the 30,988 unique assembled EST sequences (composed of 12,322 contigs and 18,666 singletons), the authors believe they have sampled roughly 92% of the S. mansoni transcriptome. These calculations suggest that approximately 12,880 distinctive S. mansoni open reading frames (ORFs) are reported in the article. As 13,131 unique clustered gene sets, representing approximately 93% gene coverage, were also identified in the S. japonicum report , similar expressed genomic complements are predicted for the two species.
The number of ESTs with significant database homology may also be similar in the two species. The S. japonicum project  reported that 65% of identified ESTs had significant database similarity to sequences already in GenBank, compared with 45% reported by the S. mansoni project . Distinct BLASTx cut-off Expect (E) values (E ≤ 10-6 for S. mansoni and E ≤ 10-3 for S. japonicum) were used in the two studies for assigning sequence similarity and are therefore likely to be responsible for this apparent difference. Taken together, the observations indicate that a significant proportion of each species' genome (approximately 50%) is schistosome-specific and may be adapted for gene expression related to parasitism. The proportion of these 'schistosome-associated genes' shared between the two species is currently unknown, but careful comparative analyses will undoubtedly reveal much about common schistosome adaptations to parasitic lifestyles. Phylogenetic analyses of both species (using highly conserved genes) indicate an early and independent divergence of schistosomes from other metazoans, which may explain the high fraction of these ESTs that display no significant database match in GenBank. It is clear that the addition of these new schistosome sequences to the public databases will contribute substantially to our understanding of evolution and the parasitic lifestyle.
The diversity of expressed Schistosoma genes
Functional classification of both parasites' genomic complement, using Interpro , to identify protein motifs, and Gene Ontology , to describe the cellular processes, predicts that the genetic machinery responsible for most cellular and physiological systems was established long before the divergence of the Platyhelminths. Although gene products associated with diverse biological functions - such as cell adhesion/tissue structure, antero-posterior axis differentiation, dorso-ventral patterning, epithelial interactions, motility, nervous system development and signaling - are present in both S. mansoni and S. japonicum, transcripts involved in metabolism and catabolism were among the most frequently characterized. The identification of these highly abundant transcripts strongly supports classical biochemical studies that have suggested that schistosomes may be completely dependent on host molecules (lipids, proteins and nucleic acids) for catabolically released energy and for the building blocks used during parasite development .
Some of the most interesting newly identified transcripts are those related to development, gender and host-parasite interactions. These transcripts may provide important insights into the evolution of morphologically distinct genders , the complete dependence of female parasites on male contact for sexual maturation [14, 15], and the mechanisms by which schistosomes evade  or modulate [17, 18, 19, 20] host immune responses. New information of this sort will undoubtedly push forward disease intervention and parasite-control strategies. Similarly, the use of comparative genomic strategies to determine the level of these transcripts in hermaphroditic trematodes should offer insights into the evolution of dioecy in this group of organisms. For example, the identification of orthologs of Caenorhabditis elegans sex-determination genes (fox-1, mog-1, mog-4, tra-2, fem-1, and mag-1) in S. mansoni , and the classification of definitive-host associated X- and Y-linked orthologs in S. japonicum , implicate these transcripts in schistosome sexual-development pathways.
There are schistosome ESTs showing sequence similarity to components of the endocrine system (for example, homologs of the insulin receptor, insulin-like growth factor 1 receptor and insulin signaling-pathway proteins), and of the immune system (for example, homologs of cytokines, cytokine-like proteins, major histocompatibility complex and allergens); these similarities strongly suggest that the parasites actively interact with host biochemical and defense mechanisms, rather than merely evading them. Modulation of host biomolecules and cellular systems has been documented [17, 18, 19, 20, 21], and the identification of novel ESTs that may function in an analogous manner points to further complex, as yet undiscovered, interactions between schistosomes and their hosts.
The schistosome transcriptome also contains EST homologs of stress-response proteins (novel heat-shock proteins, anti-oxidant enzymes, Daf16 and Toll-like molecules) and longevity-associated proteins (for example Sir-2.1, Sir-2.2, Sir-2.5, Sir-2.6 and Sir-2.7) suggesting that there are multiple genetic programs to ensure parasite survival. For example, if parasite immunomodulatory molecules fail to prevent the host's immune system from recognizing the parasite and activating a response, parasite stress-response transcripts may be induced to counteract the host immune-effector mechanisms. This has previously been proposed for the antioxidant enzymes glutathione peroxidase, superoxide dismutase and glutathione-S-transferase in S. mansoni . Similarly, if the schistosome is prevented from using its host's endocrine system, the parasite may temporarily induce genetic programs associated with longevity. Further characterization of these and other transcripts associated with hormone signaling, neurotransmission and neurogenesis present excellent opportunities to investigate novel anti-schistosome chemotherapeutic strategies. For example, the schistosome has homologs of progesterone receptor membrane components, small androgen receptor-interacting proteins, retinoic acid receptors, thyroid hormone receptor family members and progestin-induced proteins that may have endocrine function; and homologs of the neuropeptide Y2 receptor, nicotinic acetylcholine receptor, glutamate receptor, serotonin receptor, muscarinic acetylcholine receptor, Notch receptor and calcium channels that may have neuronal functions. Surprisingly, very few G-protein-coupled-receptor paralogs (rhodopsin, glutamate, serotonin, and muscarinic acetylcholine receptors) were reported in either transcriptome, suggesting that schistosomes may have a limited repertoire and may instead rely on other mechanisms to interact with environmental or self stimuli. Functional studies of these and other parasite molecules intimately involved in host-parasite interactions should shed light on the elegant schistosome survival strategies that enable them to live for years in the bloodstreams of their definitive hosts .
The future of schistosome post-genomic studies
Areas in which Schistosoma transcriptome information will be applied during the post-genomic era
Fabrication of whole transcriptome DNA microarrays
Resource for expression-profiling experiments, for example, across life-stages, between genders and so on
Construction of RNA interference (RNAi) libraries
Resource for the functional characterization of each deposited transcript
Initiation of whole-genome sequencing projects
Blueprint/template resource for initiation/completion of whole-genome sequencing projects
Characterization of proteome
Blueprint/template resource for identification of differentially expressed proteins, for example, across life-stages, between genders and so on
Investigation of evolutionary relationship amongst the metazoa
Resource for advanced phylogenetic studies
Identification of orthologous transcripts within or outside the genera
Resource for comparative genomic studies
The recent detailed transcriptome data will also provide an integral framework for the construction and completion of a full schistosome genomic map, which is currently underway at The Institute for Genomic Research (USA) and the Wellcome Trust Sanger Institute (UK), where it is expected that eightfold (8X) coverage of the S. mansoni genome will soon be obtained . This will ultimately open the path to previously unknown and valuable information about the genomes of a relatively under-studied, but biologically diverse and fascinating, phylum that contains species causing some of the world's most important and obdurate parasitic diseases.
We thank Rhian Hayward and Jennifer Fitzpatrick for critical editorial comments.
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