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

Abstract Background Five 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. Results After 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. Conclusion SAGE 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.

Analysis of Schistosoma mansoni genes shared with Deuterostomia and with possible roles in host interactions

Abstract Background: Schistosoma mansoni is a blood helminth parasite that causes schistosomiasis, a disease that affects 200 million people in the world. Many orthologs of known mammalian genes have been discovered in this parasite and evidence is accumulating that some of these genes encode proteins linked to signaling pathways in the parasite that appear to be involved with growth or development, suggesting a complex co-evolutionary process. Results: In this work we found 427 genes conserved in the Deuterostomia group that have orthologs in S. mansoni and no members in any nematodes and insects so far sequenced. Among these genes we have identified Insulin Induced Gene (INSIG), Interferon Regulatory Factor (IRF) and vasohibin orthologs, known to be involved in mammals in mevalonate metabolism, immune response and angiogenesis control, respectively. We have chosen these three genes for a more detailed characterization, which included extension of their cloned messages to obtain full-length sequences. Interestingly, SmINSIG showed a 10-fold higher expression in adult females as opposed to males, in accordance with its possible role in regulating egg production. SmIRF has a DNA binding domain, a tryptophan-rich N-terminal region and several predicted phosphorylation sites, usually important for IRF activity. Fourteen different alternatively spliced forms of the S. mansoni vasohibin (SmVASL) gene were detected that encode seven different protein isoforms including one with a complete C-terminal end, and other isoforms with shorter C-terminal portions. Using S. mansoni homologs, we have employed a parsimonious rationale to compute the total gene losses/gains in nematodes, arthropods and deuterostomes under either the Coelomata or the Ecdysozoa evolutionary hypotheses; our results show a lower losses/gains number under the latter hypothesis. Conclusion: The genes discussed which are conserved between S. mansoni and deuterostomes, probably have an ancient origin and were lost in Ecdysozoa, being still present in Lophotrochozoa. Given their known functions in Deuterostomia, it is possible that some of them have been co-opted to perform functions related (directly or indirectly) to host adaptation or interaction with host signaling processes.

On the three-finger protein domain fold and CD59-like proteins in Schistosoma mansoni

Background: It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy. Methodology/Principal Findings: Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation. Conclusions: Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.

Rapid appearance and asymmetric distribution of glucose transporter SGTP4 at the apical surface of intramammalian-stage Schistosoma mansoni.

Adult Schistosoma mansoni blood flukes reside in the mesenteric veins of their vertebrate hosts, where they absorb immense quantities of glucose through their tegument by facilitated diffusion. Previously, we obtained S. mansoni cDNAs encoding facilitated-diffusion schistosome glucose transporter proteins 1 and 4 (SGTP1 and SGTP4) and localized SGTP1 to the basal membranes of the tegument and the underlying muscle. In this study, we characterize the expression and localization of SGTP4 during the schistosome life cycle. Antibodies specific to SGTP4 appear to stain only the double-bilayer, apical membranes of the adult parasite tegument, revealing an asymmetric distribution relative to the basal transporter SGTP1. On living worms, SGTP4 is available to surface biotinylation, suggesting that it is exposed at the hose-parasite interface. SGTP4 is detected shortly after the transformation of free-living, infectious cercariae into schistosomula and coincides with the appearance of the double membrane. Within 15 min after transformation, anti-SGTP4 staining produces a bright, patchy distribution at the surface of schistosomula, which becomes contiguous over the entire surface of the schistosomula by 24 hr after transformation. SGTP4 is not detected in earlier developmental stages (eggs, sporocysts, and cercariae) that do not possess the specialized double membrane. Thus, SGTP4 appears to be expressed only in the mammalian stages of the parasite's life cycle and specifically localized within the host-interactive, apical membranes of the tegument.

A Schistosoma mansoni fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine.

Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel beta-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSm14, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively.

In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni

BACKGROUND: Schistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths.

METHODS: We have cloned one gene sequence from S. mansoni, Smp_147730 (SmKI-1) which is coded for single domain Kunitz type protease inhibitor, E. coli-expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1.

RESULTS: SmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.

CONCLUSIONS: We have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.

Haem uptake is essential for egg production in the haematophagous blood fluke of humans, Schistosoma mansoni

Schistosomes ingest host erythrocytes, liberating large quantities of haem. Despite its toxicity, haem is an essential factor for numerous biological reactions, and may be an important iron source for these helminths. We used a fluorescence haem analogue, palladium mesoporphyrin, to investigate pathways of haem acquisition, and showed that palladium mesoporphyrin accumulates in the vitellaria (eggshell precursor glands) and ovary of female Schistosoma mansoni. Furthermore, incubation of adult females in 10-100 μm cyclosporin A (IC50 = 2.3 μm) inhibits the uptake of palladium mesoporphyrin to these tissues, with tenfold reductions in fluorescence intensity of the ovary. In vitro exposure to cyclosporin A resulted in significant perturbation of egg production, reducing egg output from 34 eggs per female to 5.7 eggs per female over the incubation period, and retardation of egg development. We characterized a S. mansoni homologue of the haem-responsive genes of Caenorhabditis elegans. The gene (Smhrg-1) encodes a protein with a molecular weight of approximately 17 kDa. SmHRG-1 was able to rescue growth in haem transport-deficient HEM1Δ yeast. Transcriptional suppression of Smhrg-1 in adult S. mansoni worms resulted in significant delay in egg maturation, with 47% of eggs from transcriptionally suppressed worms being identified as immature compared with only 27% of eggs laid by control worms treated with firefly luciferase. Our findings indicate the presence of transmembrane haem transporters in schistosomes, with a high abundance of these molecules being present in tissues involved in oogenesis.

Transcriptional profiling of the oesophageal gland region of male worms of Schistosoma mansoni

The intestinal tract of schistosomes opens at the mouth and leads into the foregut or oesophageal region that is lined with syncytium continuous with the apical cytoplasm of the tegument. The oesophagus is surrounded by a specialised gland, the oesophageal gland. This gland releases materials into the lumen of the oesophagus and the region is thought to initiate the lysis of erythrocytes and neutralisation of immune effectors of the host. The oesophageal region is present in the early invasive schistosomulum, a stage potentially targetable by anti-schistosome vaccines. We used a 44k oligonucleotide microarray to identify highly up-regulated genes in microdissected frozen sections of the oesophageal gland of male worms of S. mansoni. We show that 122 genes were up-regulated 2-fold or higher in the oesophageal gland compared with a whole male worm tissue control. The enriched genes included several associated with lipid metabolism and transmembrane transport as well as some micro-exon genes. Since the oesophageal gland is important in the initiation of digestion and the fact that it develops early after invasion of the mammalian host, further study of selected highly up-regulated functionally important genes in this tissue may reveal new anti-schistosome intervention targets for schistosomiasis control.

Schistosomiasis mansoni in an area of low transmission: II. Risk factors for infection

Risk factors for Schistosoma mansoni infection were identified using a 1:1 matched case-control design. The work was conducted in the municipality of Pedro de Toledo, São Paulo State, Brazil, an area where the snail host is Biomphalaria tenagophila. Information on water contact patterns, knowledge, attitudes and pratices (kap), socioeconomic and sanitary conditions were obtained by mean of questionnaires. The crude odds ratio estimates and the adjusted odds ratio estimates using the logistic regression model are presented. Most of the examined individuals admitted recent water contacts (90.6% of the cases). The most frequent reason for contact was swimming, playing and fishing and the preferential site of contact was the river. According to the logistic regression technique, the main risk factors for infection were: a) water contact through swimming, playing and fishing; b) fording; c) bad hygiene. We concluded that recreational activities are the main reasons for schistosomiasis transmission in Pedro de Toledo and leisure alternatives should be offered to the local population.

Very low prevalence of hepatitis C virus infection in rural communities of northeastern Brazil with a high prevalence of schistosomiasis mansoni

The association of hepatitis C virus infection and the hepatosplenic form of schistosomiasis mansoni has been claimed to result in the concomitant evolution of the two pathologies, with a poor prognosis due to aggravated liver disease. Recently, however, some authors have begun to reject the hypothesis of a higher susceptibility of hepatosplenic schistosomal patients to HCV. The aim of the present transverse study carried out between July and August 1990 was to determine the possible association between SM and HCV markers in residents of Catolândia, Bahia State. Anti-HCV markers were assayed by ELISA-II and RIBA-II in serum samples obtained from 1,228 residents (85.8%). The anti-HCV antibody (ELISA-II) was positive in six (0.5%) individuals, eight (0.6%) cases were inconclusive and 1,214 (98.9%) were negative. However, only in one ELISA-positive serum sample (0.08%) were antibodies confirmed by RIBA-II, while two other samples assayed by RIBA-II were indeterminate. These three patients presented the hepatointestinal form of SM during the follow-up period (1976 to 1996). In conclusion, no association was observed between HCV and SM in the endemic area studied, especially among patients with the hepatosplenic form of the disease.

Milky spots reactions to schistosomal mansoni infection

Milky spots (MS), considered by the authors as a Coelomatic Lympho-myelopoietic Organ (CLMO), present a strong reactivity during experimental schistosomal mansoni infection, characterized by an increase of lymphocytes, macrophages, plasmocytes, mast cells, neutrophils and expression of eosinophil metaplasia. Intraperitoneal injection of purified Schistosoma mansoni (Sm) eggs provoked a rise in the number and size of MS, which developed the sessile marginal and pedunculated types. The authors conclude that egg antigens are, at least partially, responsible for MS reactivity during Sm infection.

Effects of Schistosomal mansoni infection on Calomys callosus coelom-associated lymphomyeloid tissue (milky spots)

Calomys callosus Rengger, 1830 (Rodentia: Cricetidae) is a mouse-like South American wild rodent, which is permissive to Schistosoma mansoni infection. In this paper we studied the effect of schistosomal infection in C. callosus mesenteric and omental milky spots (MS), subsidiary foci of coelom-associated lymphomyeloid tissue (CALT), during the acute, transitional (acute to chronic), and chronic phases of the infection. MS were morphologically analyzed by histological methods, using brigthfield and confocal laser scanning microscopies. The MS of infected animals were mainly of lymphomyelocytic (42 to 90 days) and lymphoplasmacytic (160 days of infection) types and showed frequent presence of lymphoid follicles with germinal centers, plasmacytogenesis and plasmacytosis, mastocytosis, megakaryopoiesis, erythropoiesis and less pronounced eosinopoiesis. These results indicate that MS are a preferential site of germinal-center-dependent and independent plasmacytogenesis, and a bone marrow-like organ, committed with various cellular lineages. The consequence of C. callosus MS reactivity for schistosomal infection is still unknown and is under investigation.