Ricardo DeMarco

The genome of the blood fluke Schistosoma mansoni

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

Schistosomes--proteomics studies for potential novel vaccines and drug targets.

Schistosomiasis is a major health problem and, despite decades of research, only one effective drug, Praziquantel is currently available. Recent expansion of sequence databases on Schistosoma mansoni and S. japonicum has permitted a wealth of novel proteomic studies on several aspects of the organization and development of the parasite in the human host. This unprecedented accumulation of molecular data is allowing a more rational approach to propose drug targets and vaccine candidates, such as proteins located at the parasite surface. Successful preliminary trials of two vaccine candidates that have been detected at the parasite surface by proteomics give grounds for believing that such an approach may provide a fresh start for the field.

Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts

Schistosoma mansoni is a well-adapted blood-dwelling parasitic helminth, persisting for decades in its human host despite being continually exposed to potential immune attack. Here, we describe in detail micro-exon genes (MEG) in S. mansoni, some present in multiple copies, which represent a novel molecular system for creating protein variation through the alternate splicing of short (< or =36 bp) symmetric exons organized in tandem. Analysis of three closely related copies of one MEG family allowed us to trace several evolutionary events and propose a mechanism for micro-exon generation and diversification. Microarray experiments show that the majority of MEGs are up-regulated in life cycle stages associated with establishment in the mammalian host after skin penetration. Sequencing of RT-PCR products allowed the description of several alternate splice forms of micro-exon genes, highlighting the potential use of these transcripts to generate a complex pool of protein variants. We obtained direct evidence for the existence of such pools by proteomic analysis of secretions from migrating schistosomula and mature eggs. Whole-mount in situ hybridization and immunolocalization showed that MEG transcripts and proteins were restricted to glands or epithelia exposed to the external environment. The ability of schistosomes to produce a complex pool of variant proteins aligns them with the other major groups of blood parasites, but using a completely different mechanism. We believe that our data open a new chapter in the study of immune evasion by schistosomes, and their ability to generate variant proteins could represent a significant obstacle to vaccine development.

Saci-1, -2, and -3 and Perere, Four Novel Retrotransposons with High Transcriptional Activities from the Human Parasite Schistosoma mansoni

ABSTRACT Using the data set of 180,000 expressed sequence tags (ESTs) of the blood fluke Schistosoma mansoni generated recently by our group, we identified three novel long-terminal-repeat (LTR)- and one novel non-LTR-expressed retrotransposon, named Saci-1, -2, and -3 and Perere, respectively. Full-length sequences were reconstructed from ESTs and have deduced open reading frames (ORFs) with several uncorrupted features, characterizing them as possible active retrotransposons of different known transposon families. Alignment of reconstructed sequences to available preliminary genome sequence data confirmed the overall structure of the transposons. The frequency of sequenced transposon transcripts in cercariae was 14% of all transcripts from that stage, twofold higher than that in schistosomula and three- to fourfold higher than that in adults, eggs, miracidia, and germ balls. We show by Southern blot analysis, by EST annotation and tallying, and by counting transposon tags from a Social Analysis of Gene Expression library, that the four novel retrotransposons exhibit a 10- to 30-fold lower copy number in the genome and a 4- to 200-fold-higher transcriptional rate per copy than the four previously described S. mansoni retrotransposons. Such differences lead us to hypothesize that there are two different populations of retrotransposons in S. mansoni genome, occupying different niches in its ecology. Examples of retrotransposon fragment inserts were found into the 5′ and 3′ untranslated regions of four different S. mansoni target gene transcripts. The data presented here suggest a role for these elements in the dynamics of this complex human parasite genome.

Influence of ecto-nucleoside triphosphate diphosphohydrolase activity on Trypanosoma cruzi infectivity and virulence.

Background The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. Methodology/Principal Findings We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. Conclusions/Significance Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

Molecular characterization and immunolocalization of Schistosoma mansoni ATP-diphosphohydrolase

Schistosoma mansoni, a human parasite that constitutes a major health problem in developing countries, escapes from host defenses and survives in the human bloodstream. Here, we report the cloning of a S. mansoni ATP-diphosphohydrolase ortholog (SmATPDase1). Southern blots indicated that in S. mansoni it is a single-copy gene. RT-PCR revealed that SmATPDase1 is expressed in five stages of the parasite life cycle, namely cercaria, schistosomula, adults, eggs, and miracidia. Using confocal microscopy, SmATPDase1 protein was immunolocalized on the external surface in all stages, except eggs, being conspicuously present in adults. ATPDase, which is present on the outer surface of endothelial cells lining human blood vessels, has been implicated in thromboregulation by promoting ADP hydrolysis and inhibition of platelet aggregation. The presence of an ATPDase ortholog on the surface of S. mansoni suggests that the enzyme might play a role in the escape from host defenses that would involve platelet activation.

A quantitative view of the transcriptome of Schistosoma mansoni adult-worms using SAGE

BackgroundFive species of the genus Schistosoma, a parasitic trematode flatworm, are causative agents of Schistosomiasis, a disease that is endemic in a large number of developing countries, affecting millions of patients around the world. By using SAGE (Serial Analysis of Gene Expression) we describe here the first large-scale quantitative analysis of the Schistosoma mansoni transcriptome, one of the most epidemiologically relevant species of this genus.ResultsAfter extracting mRNA from pooled male and female adult-worms, a SAGE library was constructed and sequenced, generating 68,238 tags that covered more than 6,000 genes expressed in this developmental stage. An analysis of the ordered tag-list shows the genes of F10 eggshell protein, pol-polyprotein, HSP86, 14-3-3 and a transcript yet to be identified to be the five top most abundant genes in pooled adult worms. Whereas only 8% of the 100 most abundant tags found in adult worms of S. mansoni could not be assigned to transcripts of this parasite, 46.9% of the total ditags could not be mapped, demonstrating that the 3 sequence of most of the rarest transcripts are still to be identified. Mapping of our SAGE tags to S. mansoni genes suggested the occurrence of alternative-polyadenylation in at least 13 gene transcripts. Most of these events seem to shorten the 3 UTR of the mRNAs, which may have consequences over their stability and regulation.ConclusionSAGE revealed the frequency of expression of the majority of the S. mansoni genes. Transcriptome data suggests that alternative polyadenylation is likely to be used in the control of mRNA stability in this organism. When transcriptome was compared with the proteomic data available, we observed a correlation of about 50%, suggesting that both transcriptional and post-transcriptional regulation are important for determining protein abundance in S. mansoni. The generation of SAGE tags from other life-cycle stages should contribute to reveal the dynamics of gene expression in this important parasite.

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

BackgroundThe CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism.ResultsWe describe a novel S. mansoni transposon named SmTRC1, for S. mansoniT ransposon R elated to C ACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence.ConclusionThe DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

Schistosoma mansoni Egg, Adult Male and Female Comparative Gene Expression Analysis and Identification of Novel Genes by RNA-Seq

Background Schistosomiasis is one of the most prevalent parasitic diseases worldwide and is a public health problem. Schistosoma mansoni is the most widespread species responsible for schistosomiasis in the Americas, Middle East and Africa. Adult female worms (mated to males) release eggs in the hepatic portal vasculature and are the principal cause of morbidity. Comparative separate transcriptomes of female and male adult worms were previously assessed with using microarrays and Serial Analysis of Gene Expression (SAGE), thus limiting the possibility of finding novel genes. Moreover, the egg transcriptome was analyzed only once with limited bacterially cloned cDNA libraries. Methodology/Principal findings To compare the gene expression of S. mansoni eggs, females, and males, we performed RNA-Seq on these three parasite forms using 454/Roche technology and reconstructed the transcriptome using Trinity de novo assembly. The resulting contigs were mapped to the genome and were cross-referenced with predicted Smp genes and H3K4me3 ChIP-Seq public data. For the first time, we obtained separate, unbiased gene expression profiles for S. mansoni eggs and female and male adult worms, identifying enriched biological processes and specific enriched functions for each of the three parasite forms. Transcripts with no match to predicted genes were analyzed for their protein-coding potential and the presence of an encoded conserved protein domain. A set of 232 novel protein-coding genes with putative functions related to reproduction, metabolism, and cell biogenesis was detected, which contributes to the understanding of parasite biology. Conclusions/Significance Large-scale RNA-Seq analysis using de novo assembly associated with genome-wide information for histone marks in the vicinity of gene models constitutes a new approach to transcriptome analysis that has not yet been explored in schistosomes. Importantly, all data have been consolidated into a UCSC Genome Browser search- and download-tool (http://schistosoma.usp.br/). This database provides new ways to explore the schistosome genome and transcriptome and will facilitate molecular research on this important parasite.