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The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine., To date there is only one single drug with modest efficacy and no vaccine available to protect from schistosomiasis. Here, Amaral et al. characterize the self-cure process of rhesus macaques following primary infection and secondary challenge with Schistosoma mansoni to inform future vaccine development studies.

The unique multilaminate appearance of the tegument surface of schistosomes was first described in 1973, in one of the earliest volumes of the International Journal for Parasitology. The present review, published almost 50 years later, traces the development of our knowledge of the tegument, starting with those earliest cytological advances, particularly the surface plasma membrane-membranocalyx complex, through an era of protein discovery to the modern age of protein characterization, aided by proteomics. More recently, analysis of single cell transcriptomes of schistosomes is providing insight into the organization of the cell bodies that support the surface syncytium. Our understanding of the tegument, notably the nature of the proteins present within the plasma membrane and membranocalyx, has provided insights into how the schistosomes interact with their hosts but many aspects of how the tegument functions remain unanswered. Among the unresolved aspects are those concerned with maintenance and renewal of the surface membrane complex, and whether surface proteins and membrane components are recycled. Current controversies arising from investigations about whether the tegument is a source of extracellular vesicles during parasitism, and if it is covered with glycolytic enzymes, are evaluated in the light of cytological and proteomic knowledge of the layer.

The radiation-attenuated cercarial vaccine remains the gold standard for the induction of protective immunity againstSchistosoma mansoni. Furthermore, the protection can be passively transferred to naïve recipient mice from multiply vaccinated donors, especially IFNgR KO mice. We have used such sera versus day 28 infection serum, to screen peptide arrays and identify likely epitopes that mediate the protection. The arrays encompassed 55 secreted or exposed proteins from the alimentary tract and tegument, the principal interfaces with the host bloodstream. The proteins were printed onto glass slides as overlapping 15mer peptides, reacted with primary and secondary antibodies, and reactive regions detected using an Agilent array scanner. Pep Slide Analyzer software provided a numerical value above background for each peptide from which an aggregate score could be derived for a putative epitope. The reactive regions of 26 proteins were mapped onto crystal structures using the CCP4 molecular graphics, to aid selection of peptides with the greatest accessibility and reactivity, prioritizing vaccine over infection serum. A further eight MEG proteins were mapped to regions conserved between family members. The result is a list of priority peptides from 44 proteins for further investigation in multiepitope vaccine constructs and as targets of monoclonal antibodies.

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Only with the completion of the life cycles of Fasciola hepatica in 1883 and 30 years later those of Schistosoma japonicum (1913), Schistosoma haematobium and Schistosoma mansoni (1915) did research on schistosomiasis really get underway. One of the first papers by Cawston in 1918, describing attempts to establish the means of transmission of S. haematobium in Natal, South Africa, forms the historical perspective against which to judge where we are now. Molecular biology techniques have produced a much better definition of the complexity of the schistosome species and their snail hosts, but also revealed the extent of hybridization between human and animal schistosomes that may impact on parasite adaptability. While diagnostics have greatly improved, the ability to detect single worm pair infections routinely, still falls short of its goal. The introduction of praziquantel ~1982 has revolutionized the treatment of infected individuals and led directly to the mass drug administration programmes. In turn, the severe pathological consequences of high worm burdens have been minimized, and for S. haematobium infections the incidence of associated squamous cell carcinoma has been reduced. In comparison, the development of effective vaccines has yet to come to fruition. The elimination of schistosomiasis japonica from Japan shows what is possible, using multiple lines of approach, but the clear and present danger is that the whole edifice of schistosome control is balanced on the monotherapy of praziquantel, and the development of drug resistance could topple that.

Schistosomes are blood-dwelling helminth parasites that cause schistosomiasis, a debilitating disease resulting in inflammation and, in extreme cases, multiple organ damage. Major challenges to control the transmission persist, and the discovery of protective antigens remains of critical importance for vaccine development. Rhesus macaques can self-cure following schistosome infection, generating antibodies that target proteins from the tegument, gut, and esophagus, the last of which is the least investigated. We developed a dissection technique that permitted increased sensitivity in a comparative proteomics profiling of schistosome esophagus and gut. Proteome analysis of the male schistosome esophagus identified 13 proteins encoded by microexon genes (MEGs), 11 of which were uniquely located in the esophageal glands. Based on this and transcriptome information, a QconCAT was designed for the absolute quantification of selected targets. MEGs 12, 4.2, and 4.1 and venom allergen-like protein 7 were the most abundant, spanning over 245 million to 6 million copies per cell, while aspartyl protease, palmitoyl thioesterase, and galactosyl transferase were present at

Background The development of a schistosome vaccine has proved challenging but we have suggested that characterisation of the self-cure mechanism in rhesus macaques might provide a route to an effective product. The schistosome esophagus is a complex structure where blood processing is initiated by secretions from anterior and posterior glands, achieved by a mixture of ~40 unique proteins. The mechanism of self-cure in macaques involves cessation of feeding, after which worms slowly starve to death. Antibody coats the esophagus lumen and disrupts the secretory processes from the glands, potentially making their secretions ideal vaccine targets. Methodology/principal findings We have designed three peptide arrays comprising overlapping 15-mer peptides encompassing 32 esophageal gland proteins, and screened them for reactivity against 22-week infection serum from macaques versus permissive rabbit and mouse hosts. There was considerable intra- and inter-species variation in response and no obvious unique target was associated with self-cure status, which suggests that self-cure is achieved by antibodies reacting with multiple targets. Some immuno-dominant sequences/regions were evident across species, notably including: MEGs 4.1C, 4.2, and 11 (Array 1); MEG-12 and Aspartyl protease (Array 2); a Tetraspanin 1 loop and MEG-n2 (Array 3). Responses to MEGs 8.1C and 8.2C were largely confined to macaques. As proof of principle, three synthetic genes were designed, comprising several key targets from each array. One of these was expressed as a recombinant protein and used to vaccinate rabbits. Higher antibody titres were obtained to the majority of reactive regions than those elicited after prolonged infection. Conclusions/significance It is feasible to test simultaneously the additive potential of multiple esophageal proteins to induce protection by combining their most reactive regions in artificial constructs that can be used to vaccinate suitable hosts. The efficacy of the approach to disrupt esophageal function now needs to be tested by a parasite challenge.

The inadequacy of current diagnostics for the detection of low worm burdens in humans means that schistosomiasis mansoni is more widespread than previously acknowledged. With the inception of mass drug treatment programmes aimed at disease elimination and the advent of human vaccine trials, the need for more sensitive diagnostics is evident. In this review, we evaluate the merits and limitations of the principal diagnostic methods, namely detection of eggs in faeces; anti-schistosome antibodies in serum; parasite-derived proteins and glycans in serum or urine; parasite DNA in blood, faeces or urine. Only in the baboon model, where actual worm burden is determined by portal perfusion, have faecal smear and circulating antigen methods been calibrated, and shown to have thresholds of detection of 10–19 worm pairs. There is scope for improvement in all the four methods of detection, e.g. the identification of single targets for host antibodies to improve the specificity of enzyme linked immunosorbent assay. Despite recent advances in the definition of the schistosome secretome, there have been no comprehensive biomarker investigations of parasite products in the urine of infected patients. Certainly, the admirable goal of eliminating schistosomiasis will not be achieved unless individuals with low worm burdens can be diagnosed.

Baboons vaccinated with radiation-attenuated cercariae develop high levels of protection against schistosome infection, correlating to high antibody titres towards schistosome antigens with unknown molecular identity. Using a microarray consisting of glycans isolated from different life-stages of schistosomes, we studied the anti-glycan immunoglobulin (Ig) G and IgM responses in vaccinated and challenged baboons over a time course of 25 weeks. Anti-glycan IgM responses developed early after vaccination, but did not rise in response to later vaccinations. In contrast, anti-glycan IgG developed more slowly, but was boosted by all five subsequent vaccinations. High IgM and IgG levels against O-glycans and glycosphingolipid glycans of cercariae were observed. At the time of challenge, while most antibody levels decreased in the absence of vaccination, IgG towards a subset of glycans containing multiple-fucosylated motifs remained high until 6 weeks post-challenge during challenge parasite elimination, suggesting a possible role of this IgG in protection.

Schistosomes are intravenous parasiteswith ability to survive in the mammalian host for decades, using its blood as a source of nutrients.The feeding process is multistep and takes place along the worm’s alimentary tract, which comprises an (i) oral cavity opening to a short (ii) esophagus that is connected to the (iii) the gut caecum. The ultrastructureal morphologyofSchistosoma mansoniandS. japonicumhas revealed the existence of two secretory cell masses surrounding the esophagus tube, referred to as the anterior and posterior esophageal glands (antESO and postESO, respectively). We recently established that the esophageal glands have a pivotal role in the first steps of blood processing. For instance, erythrocytes and leucocytes are quickly processed along the esophagus before they are propelled to the lower parts of the intestines for further digestion nutrient uptake. We propose that incorrect functioning of alimentary tract is associated with worms death by starvation. This was first observed in the self-cure response of Rhesus macaque (Macaca mulatta), one of few known vertebratehosts capable of combating the disease through worm elimination once the infection is stablished.Classical immunoproteomics (2D-PAGE and Western blotting) has revealed potential targets in both exposed tegument and secreted gut proteins. Recently, a more detailedinvestigation using S. japonicumin the Rhesus model shed light on the possible operating mechanisms that prevent parasite feeding on blood. In addition, ultrastructural studies and immunocytochemistry on surviving worms indicated the esophageal lumen and the gland secretions as the primary targets of a potent and protective humoral immune response that ultimately disrupts the esophageal functions. Therefore, the molecular characterisation of the esophageal gland constituents is imperativeif one intends to emulate the Rhesus self-cure response for therapeutic purposes. However, this is not a trivial task aschallenges are multiple. Perhaps, the most important caveat is that both anterior and posterior parts of the oesophageal gland represent a minor fraction of the whole parasite body (or even of its head), meaning that dominant constituents, such as those derived from muscle tissues, suppress the identification of a unique set of gland products. Indeed, our recent investigation on the soluble protein composition of aS. mansonipreparation failed to detect the presence of gland products attesting for their low abundance in the whole worm. Although optimized protocols for chemical/enzymatic dissection arereported forisolation of testes and ovary of adult worms, no methodhas proved feasible for gastrodermal epithelium and the esophageal gland cells. We tackled these challenges by developing a dissection technique on worms preserved in RNAlater solution aided by an essential set of scissors and tweezers that delivers adequate precision during the procedure. The method herein described is compatible with downstream proteomic analyses using mass spectrometry and has permitted a large-scale characterisation of proteins expressed in the S. mansoni esophagus and gastrodermis. This methodology can be applied in the molecular characterisation of other schistosome organs and tissues that present a well-defined anatomic location.

Schistosomes are intravenous parasiteswith ability to survive in the mammalian host for decades, using its blood as source of nutrients.The feeding process is multistep and takes place along the worm’s alimentary tract, which comprises an (i) oral cavity opened to a short (ii) oesophagus that is connected to the (iii) gut caecum. The ultrastructure morphologyofSchistosoma mansoniandS. japonicumhas revealed the existence of two secretory cell masses surrounding the oesophagus tube, referred to as anterior and posterior oesophageal glands (antESO and postESO, respectively). We recently established that the oesophageal glands have a pivotal role in the first steps of blood processing. For instance, erythrocyte and leucocyte are quickly neutralized along the oesophagus before they are propelled to the lower parts of the intestines for further digestion nutrient uptake. We knowledge that incorrect functioning of alimentary tract is associated with worms death by starvation. This was firstly observed in the self-cure response of Rhesus macaque (Macaca mulatta), the only known vertebratecapable of combating the disease through worm elimination once the infection is stablished.Classical immunoproteomics (2D-PAGE and Western blotting) has revealed potential targets in both exposed tegument and secreted gut proteins. Recently, a more detailedinvestigation using theS. japonicumin the Rhesus model shed light on the possible operating mechanisms that prevent parasite feeding on blood. Ultrastructural studies and immunocytochemistry on surviving worms indicated the esophageal lumen and the gland secretions as the primary targets of a potent and protective humoral immune response that ultimately disrupts the oesophageal functions. Therefore, the molecular characterisation of the oesophageal gland constituents is imperativeif one intends to emulate the Rhesus self-cure response for therapeutical purposes. However, this is not a trivial task and aschallenges are multiple. Perhaps, the most important caveat is that both anterior and posterior parts of the oesophageal gland represent a minor fraction of the whole parasite body (or even of its head), meaning that dominant constituents, such as those derived from muscle tissues, suppress the identification of the unique set of gland products. Indeed, our recent investigation on the soluble protein composition of aS. mansonipreparation failed to reveal their presence attesting for their low abundance. Although optimized protocols for chemical/enzymatic dissection arereported forisolation of testes and ovary of adult worms, not a single methodhas proved feasible for gastrodermal epithelium and the oesophageal gland cells. We tackled these challenges by developing a dissection technique of male and female worms preserved in RNAlater solution that entirely compatible with downstreamRNAseq and proteomics analyses. This collection contains all methods developed by our group that have permitted the large-scale characterization of transcripts and proteins expressed in theS. mansoniandS. japonicumoesophagus and gastrodermis.

Background Adult schistosomes have a well-developed alimentary tract comprising an oral sucker around the mouth, a short esophagus and a blind ending gut. The esophagus is not simply a muscular tube for conducting blood from the mouth to gut but is divided into compartments, surrounded by anterior and posterior glands, where processing of ingested blood is initiated. Self-cure of rhesus macaques from a Schistosoma japonicum infection appears to operate by blocking the secretory functions of these glands so that the worms cease feeding and slowly starve to death. Here we use subtractive RNASeq to characterise the genes encoding the principal secretory products of S. japonicum esophageal glands, preparatory to evaluating their relevance as targets of the self-cure process. Methodology/Principal findings The heads and a small portion of the rear end of male and female S. japonicum worms were separately enriched by microdissection, for mRNA isolation and library construction. The sequence reads were then assembled de novo using Trinity and those genes enriched more than eightfold in the head preparation were subjected to detailed bioinformatics analysis. Of the 62 genes selected from the male heads, more than one third comprised MEGs encoding secreted or membrane-anchored proteins. Database searching using conserved motifs revealed that the MEG-4 and MEG-8/9 families had counterparts in the bird schistosome Trichobilharzia regenti, indicating an ancient association with blood processing. A second group of MEGs, including a MEG-26 family, encoded short peptides with amphipathic properties that most likely interact with ingested host cell membranes to destabilise them. A number of lysosomal hydrolases, two protease inhibitors, a secreted VAL and a putative natterin complete the line-up. There was surprisingly little difference between expression patterns in males and females despite the latter processing much more blood. Significance/Conclusions The mixture of approximately 40 proteins specifically secreted by the esophageal glands is responsible for initiating blood processing in the adult worm esophagus. They comprise the potential targets for the self-cure process in the rhesus macaque, and thus represent a completely new cohort of secreted proteins that can be investigated as vaccine candidates., Author summary Schistosomes are parasitic flatworms inhabiting the human bloodstream, surrounded by and feeding on humoral and cellular components of the immune system. They are normally long-lived but the rhesus macaque is able to mount a self-cure response directed against the esophageal secretions of the adult Schistosoma japonicum so that they stop feeding and slowly starve to death. The worm esophagus is a short tube connecting mouth to gut surrounded by anterior and posterior glands and our aim in this study was to identify the genes encoding the gland secretions. For this purpose we isolated the messenger RNA from both male and female worm heads and tails and obtained many millions of sequences. These were assembled into gene coding sequences using bioinformatics and then genes differentially expressed in the head region were identified by a subtraction process. We then focused on those genes encoding proteins with a leader sequence indicating their secretory status. The result is an inventory of approximately 40 genes; some encode protein binding motifs while others encode a short helix with a hydrophobic face, which may interact with host cell membranes. Genes encoding enzymes, protease inhibitors and a venom-like protein were also found. These proteins are being evaluated for their interactions with the antibodies generated by macaques during the self-cure process.

Human schistosomiasis caused by parasitic flatworms of the genus Schistosoma remains an important public health problem in spite of concerted efforts at control. An effective vaccine would be a useful addition to control strategies that currently rely on chemotherapy, but such a product is not imminent. In this review, likely causes for the lack of progress are first considered. These include the strategies used by worms to evade the immune response, concepts that have misdirected the field, an emphasis on internal antigens, and the use of the laboratory mouse for vaccine testing. On a positive note, recent investigations on self-cure by the rhesus macaque offer the most promising context for vaccine development. The identification of proteins at the parasite–host interface, especially those of the esophageal glands involved in blood processing, has provided an entirely new category of vaccine candidates that merit evaluation.

Background Human immunity to Schistosoma infection requires many years of exposure, and multiple infections and treatments to develop. Unlike humans, rhesus macaques clear an established schistosome infection naturally at the same time acquiring immunity towards re-infection. In macaques, schistosome egg production decreases after 8 weeks post-infection and by week 22, physiological impairment of the worm caused by unclarified antibody-mediated processes is observed. Since strong antibody responses have been observed against schistosome glycan antigens in human and animal infections, we here investigate if anti-glycan antibodies are associated with immunity against schistosome infections in macaques. Methods We used a microarray containing a large repertoire of glycoprotein- and glycolipid-derived glycans from different schistosome life stages to analyse anti-glycan serum IgG and IgM from S. japonicum-infected macaques during the course of infection and self-cure. We also used an in vitro schistosomula assay to investigate whether macaque sera containing anti-glycan antibodies can kill schistosomula. Conclusions/significance Antibody responses towards schistosome glycans at week 4 post-infection were dominated by IgM while IgG was high at week 8. The profound increase in IgG was observed mainly for antibodies towards a large subset of glycans that contain (multi-)fucosylated terminal GalNAcβ1-4GlcNAc (LDN), and Galβ1-4(Fucα1–3)GlcNAc (LeX) motifs. In general, glycans with a higher degree of fucosylation gave rise to stronger antibody responses than non-fucosylated glycans. Interestingly, even though many IgG and IgM responses had declined by week 22 post-infection, IgG towards O-glycans with highly fucosylated LDN motifs remained. When incubating macaque serum with schistosomula in vitro, schistosomula death was positively correlated with the duration of infection of macaques; macaque serum taken 22 weeks post-infection caused most schistosomula to die, suggesting the presence of potentially protective antibodies. We hypothesize that IgGs against highly fucosylated LDN motifs that remain when the worms deteriorate are associated with infection clearance and the resistance to re-infection in macaques., Author summary Schistosomes express many glycan antigens to which antibodies are raised by the infected host. These glycans may therefore form potential vaccine targets. Unlike humans where the disease persists chronically if not treated, schistosome-infected rhesus macaques are able to elicit a self-cure process naturally. To find out if anti-glycan responses could contribute to the natural clearance process, we followed the dynamics of anti-glycan serum antibodies in Schistosoma-infected macaques in a longitudinal study starting from the onset of infection until 22 weeks post-infection, when the macaques had eliminated most of the parasites. We found that sera of macaques taken after 22 weeks of infection contained high IgG titres towards specific schistosome glycan epitopes highly abundant on schistosome larvae. Moreover, infected macaque serum at week 22 was able to kill schistosomula in vitro. Our results suggest that anti-glycan antibodies play an important role in the self-cure process and the acquired resistance to re-infection in Schistosoma infected macaques.

This review considers whether the products of schistosomes in the mammalian host can be considered as virulence factors. These include: the cercarial secretions used in infection, those of the migrating schistosomulum, surface-exposed proteins of adult worms in the portal system and their gut vomitus in the context of immune evasion, secretions of the egg facilitating its escape from gut tissues and micro-exon gene products.

The Schistosoma mansoni Venom Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in host-pathogen interactions. Whole mount in situ hybridisation demonstrated a distinct expression pattern in oral and ventral suckers of adult worms for SmVAL6 and in the oesophageal gland for SmVAL7 transcripts, respectively. Additionally, immunocytochemistry analysis corroborated SmVAL7 expression in the oesophageal gland. Analysis of protein expression across the parasite's life cycle revealed that the SmVAL6 protein is upregulated in cercariae and adult male worms. Furthermore, SmVAL6 protein was identified by mass spectrometry in tegument fractions of adult worms. Finally, we speculate on possible functions of these two SmVALs at the host-parasite interface.

SUMMARYSince 2004 there has been a remarkable increment in our knowledge of the proteins and glycans that reside at, or are released from the surfaces of schistosomes in the mammalian host. Initial characterization of the soluble proteome permits distinctions to be made between the parasite secretome and its necrotome. The principal proteins secreted by the cercaria to gain access to the skin have been described as well as those released by migrating schistosomula. An inventory of transporters, enzymes and structural proteins has been shown to reside the tegument surface, but also immunoglobulins, complement factors and host CD44. The secreted membranocalyx that overlies the plasma membrane may contain a small number of proteins, not simply acting as physical barrier, but its lipid composition remains elusive. Analysis of worm vomitus has provided insights into blood feeding, increasing the number of known lysosomal hydrolases, and identifying a series of carrier proteins potentially involved in uptake of lipids and inorganic ions by the gut epithelium. The egg secretions that aid escape from the tissues include a mixture of MEG-2 and MEG-3 family variant proteins. The utility of identified proteins for the development of new diagnostics, and their potential as vaccines candidates is evaluated.

We designed a controlled experiment to assess the characteristics of operationally harvested wood chips across a variety of stand ages, species and soil types in the coastal plain of Georgia. A whole tree chipping crew harvested ten stands, five loblolly pine (Pinus taeda) and five slash pine (Pinus. elliotti), in the coastal plain of Georgia. Seven samples of chips were taken from each tract during harvesting from trees dispersed across the sites. Five samples were taken from whole-tree chips directly from the outfeed of the chipper, one sample represented chips made from trees pulled through a chain-flail delimber, and the final sample was from chips collected at the mill site during the off-loading of the truck. Bark represented 10–14% of the total sample weight, while foliage represented around 1.5% on average. Small but significant differences were present between the moisture (3%) and energy content (1%) of samples from the two species. Method of sample collection had a significant impact on the size distribution and composition of samples collected. Foliage levels in the sample had a substantial impact on the nutrient composition, while bark levels had a lesser impact.

This review of schistosome cell biology has a dual purpose; its intent is to alert two separate research communities to the activities of the other. Schistosomes are by far and away the best-characterised platyhelminths, due to their medical and economic importance, but seem to be almost totally ignored by researchers on the free-living lower metazoans. Equally, in their enthusiasm for the parasitic way of life, schistosome researchers seldom pay attention to the work on free-living animals that could inform their molecular investigations. The publication of transcriptomes and/or genomes for Schistosoma mansoni and Schistosoma japonicum, the sponge Archimedon, the cnidarians Nematostella and Hydra and the planarian Schmidtea provide the raw material for comparisons. Apart from interrogation of the databases for molecular similarities, there have been differences in technical approach to these lower metazoans; widespread application of whole mount in situ hybridisation to Schmidtea contrasts with the application of targeted proteomics to schistosomes. Using schistosome cell biology as the template, the key topics of cell adhesion, development, signalling pathways, nerve and muscle, and epithelia, are reviewed, where possible interspersing comparisons with the sponge, cnidarian and planarian data. The biggest jump in the evolution of cellular capabilities appears to be in the transition from a diploblast to triploblast level of organisation associated with development of a mobile and plastic body form.

SUMMARYTransposons of the Mutator superfamily have been widely described in plants, but only recently have metazoan organisms been shown to harbour them. In this work we describe novel Mutator superfamily transposons from the genomes of the human parasitesSchistosoma mansoniandS. japonicum, which we name Curupira-1 and Curupira-2. Curupira elements do not have Terminal Inverted Repeats (TIRs) at their extremities and generate Target Site Duplications (TSDs) of 9 base pairs. Curupira-2 transposons code for a conserved transposase and SWIM zinc finger domains, while Curupira-1 elements comprise these same domains plus a WRKY zinc finger. Alignment of transcript sequences from both elements back to the genomes indicates that they are subject to splicing to produce mature transcripts. Phylogenetic analyses indicate that these transposons represent a new lineage of metazoan Mutator-like elements with characteristics that are distinct from the recently described Phantom elements. Description of these novel schistosome transposons provides new insights in the evolution of transposable elements in schistosomes.

Schistosoma mansoni eggs, consisting of an ovum surrounded by nutritive vitelline cells packaged in a tanned protein shell, are produced by paired worms residing in the mesenteric veins of the human host. The vitelline cells are degraded as the larval miracidium matures, the fully developed egg either crossing the gut wall to escape the host or becoming lodged in the host's tissues where it dies and disintegrates, inducing a potentially pathological immune response. Thus, the egg is central to both the transmission of the parasite and the aetiology of the disease. Here we present the first study investigating protein turnover in the egg. We establish that the ubiquitin-proteasome pathway (UPP) changes with egg development and furthermore, that the morphological components of the fully developed egg (the miracidium and the subshell envelope) also exhibit different proteasome subunit expression profiles. We conclude that the UPP is responsible not only for degrading the vitelline cells but is also more highly developed in the envelope than in the miracidium. The envelope is involved in the defence of the miracidium and produces the proteins that the egg secretes, presumably to facilitate its escape from the host, so the UPP probably has a multi-faceted role in the egg's biology.

Tryptophan hydroxylase (TPH) is a key enzyme in the synthesis of serotonin. As a neurotransmitter, serotonin plays important physiological roles both peripherally and centrally. Here we describe the discovery of substituted triazines as a novel class of tryptophan hydroxylase inhibitors. This class of TPH inhibitors can selectively reduce serotonin levels in murine intestine after oral administration without affecting levels in the brain. These TPH inhibitors may provide novel treatments for gastrointestinal disorders associated with dysregulation of the serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.

A recombinant antigen vaccine against Schistosoma mansoni remains elusive, in part because the parasite deploys complex defensive and offensive strategies to combat immune attack. Nevertheless, research on rodent and primate models has shown that schistosomes can be defeated when appropriate responses are elicited. Acquired protection appears to involve protracted inhibition of larval migration or key molecular processes at the adult surfaces, not rapid cytolytic killing mechanisms. A successful vaccine will likely require a cocktail of antigens rather than a single recombinant protein. In addition, ways need to be found of keeping the immune system on permanent alert, either to achieve adequate inhibition of protein function in adults, or because a trickle of incoming parasites does not amplify the secondary response.

The secretome of a parasite in its definitive host can be considered to be its genome in trans, to the extent that secreted products encoded by the parasite fulfill their function in the host milieu. The 'extended phenotype' of the filarial parasite, Brugia malayi, is of particular interest because of the evidence that infection results in potent down-modulation of the host immune response. We collected B. malayi 'excretory-secretory' (BES) proteins from adult parasites and using a combination of shotgun LC-MS/MS and 2D gel electrophoresis, identified 80 B. malayi and two host proteins in BES, of which 31 (38%) were detectable in whole worm extract (BmA). Products which were enriched in BES relative to BmA included phosphatidylethanolamine-binding protein (PEB), leucyl aminopeptidase (LAP, homologue of ES-62 from the related filaria Acanthocheilonema viteae), N-acetylglucosaminyltransferase (GlcNAcT) and galectin-1, in addition to the previously described major surface glycoprotein, glutathione peroxidase (gp29, GPX-1) and the cytokine homologue macrophage migration inhibitory factor (MIF-1). One of the most abundant released proteins was triose phosphate isomerase (TPI), yet many other glycolytic enzymes (such as aldolase and GAPDH) were found only in the somatic extract. Among the more prominent novel products identified in BES were a set of 11 small transthyretin-like proteins, and three glutamine-rich-repeat mucin-like proteins. Notably, no evidence was found of any secreted protein corresponding to the genome of the Wolbachia endosymbiont present in B. malayi. Western blotting with anti-phosphorylcholine (PC) monoclonal antibody identified that GlcNAcT, and not the ES-62 homologue, is the major PC-bearing protein in BES, while probing with human filariasis sera showed preferential reactivity to galectin-1 and to processed forms of myotactin. Overall, this analysis demonstrates selective release of a suite of newly identified proteins not previously suspected to be involved at the host-parasite interface, and provides important new perspectives on the biology of the filarial parasite.

5-Hydroxytryptamine (serotonin) (5-HT) is a neurotransmitter with both central and peripheral functions, including the modulation of mood, appetite, hemodynamics, gastrointestinal (GI) sensation, secretion, and motility. Its synthesis is initiated by the enzyme tryptophan hydroxylase (TPH). Two isoforms of TPH have been discovered: TPH1, primarily expressed in the enterochromaffin cells of the gastrointestinal tract, and TPH2, expressed exclusively in neuronal cells. Mice lacking Tph1 contain little to no 5-HT in the blood and GI tract while maintaining normal levels in the brain. Because GI 5-HT is known to play important roles in normal and pathophysiology, we set out to discover and characterize novel compounds that selectively inhibit biosynthesis of GI 5-HT. Here, we describe two of a series of these inhibitors that are potent for TPH activity both in biochemical and cell-based assays. This class of compounds has unique properties with respect to pharmacokinetic and pharmacodynamic effects on GI serotonin production. Similar to the Tph1 knockout results, these TPH inhibitors have the ability to selectively reduce 5-HT levels in the murine GI tract without affecting brain 5-HT levels. In addition, administration of these compounds in a ferret model of chemotherapy-induced emesis caused modest reductions of intestinal serotonin levels and a decreased emetic response. These findings suggest that GI-specific TPH inhibitors may provide novel treatments for various gastrointestinal disorders associated with dysregulation of the GI serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.

Recent work has implicated schistosome albumin as part of a mechanism for neutralizing the oxidative assault by host immune defenses and suggested that the gene had been acquired by horizontal transfer from the mammalian host. In the course of proteomic analyses of Schistosoma mansoni adult worm vomitus and eggs recovered from mice, we identified numerous peptides, largely derived from murine rather than parasite albumin. We therefore conjectured that the supposed S. mansoni albumin sequence deposited on GenBank might be the result of contamination rather than horizontal gene transfer. Based on phylogenetic analysis the most likely source was the Syrian (golden) hamster Mesocricetus auratus. Proteomic analysis of Syrian hamster albumin generated peptide identities to S. mansoni as the top hit, with a high ion score >1,500 and 63% coverage of the translated cDNA sequence. RT-PCR using specific primers permitted amplification of the M. auratus albumin transcript, which is identical to the deposited S. mansoni albumin sequence. PCR amplification of a fragment of the M. auratus albumin gene from genomic DNA suggests a homologous structure to the Mus musculus albumin gene. We were unable to find the S. mansoni albumin gene sequence by in silico searching on either version 3 of the S. mansoni genome assembly or the >3 million shotgun DNA reads. Finally, Southern blotting detected the albumin gene in M. auratus but not in S. mansoni genomic DNA, even when the latter was present in a 10-fold excess. Collectively, our data make the strongest case that the schistosome albumin protein described in previous reports is of host origin and all nucleotide-derived data are the result of contamination with host material. By analogy, we suggest that other reported examples of horizontal gene transfer to schistosomes might similarly be explained by complementary/genomic DNA contamination.

The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries. Glycoconjugates, and in particular the carbohydrate component of these products, represent the main immunogenic challenge to the host and could therefore represent one of the crucial determinants for successful parasite establishment. Here we report a comparative glycomics analysis of the N- and O-glycans derived from glycoproteins present in S. mansoni egg (egg-secreted protein) and cercarial (0-3-h released protein) secretions by a combination of mass spectrometric techniques. Our results show that S. mansoni secrete glycoproteins with glycosylation patterns that are complex and stage-specific. Cercarial stage secretions were dominated by N-glycans that were core-xylosylated, whereas N-glycans from egg secretions were predominantly core-difucosylated. O-Glycan core structures from cercarial secretions primarily consisted of the core sequence Galbeta1--3(Galbeta1--6)GalNAc, whereas egg-secreted O-glycans carried the mucin-type core 1 (Galbeta1--3GalNAc) and 2 (Galbeta1--3(GlcNAcbeta1--6)GalNAc) structures. Additionally we identified a novel O-glycan core in both secretions in which a Gal residue is linked to the protein. Terminal structures of N- and O-glycans contained high levels of fucose and include stage-specific structures. These glycan structures identified in S. mansoni secretions are potentially antigenic motifs and ligands for carbohydrate-binding proteins of the host immune system.

Proteasomes are molecular machines found in virtually all cells that provide one of the mechanisms for protein turnover. We have analysed the 20S proteasome of Schistosoma mansoni, the first multimeric complex isolated from this helminth parasite. Three chromatographic steps were employed to yield a highly homogeneous preparation. 2-DE of the purified complex revealed 58 spots, of which 46 could be assigned either an alpha or a beta proteasome signature by MS. Most of the 14 transcripts (7alpha and 7beta) encoded by the parasite genome were represented by multiple spots and we suggest that this diversity is due to PTMs of subunits. For most of the isoforms, variations in pI predominated although alterations in mass were also observed. 2-DE separations of extracts from infective cercariae and blood-dwelling adult worms probed by Western blotting, using a human anti-alpha subunit antibody, revealed different patterns of reactivity, most probably in alpha3 and alpha6 subunits, on the basis of sequence conservation. This difference was rapidly lost following transformation of the cercaria to the skin schistosomulum stage, suggesting that changes in the proteasome structure, likely caused by the introduction of a new set of PTMs, precede remodelling of the parasite body prior to intravascular migration.

The recent release of version 3 of the Schistosoma mansoni genome assembly has made a wealth of information available to researchers. Here, progress made in schistosome genomics and post-genomics is considered. The current status of knowledge about the genome, transcriptome, proteome, glycome and immunome is summarized and recent publications briefly reviewed. The prospects for advances in understanding schistosome biology are highlighted. Most importantly, the limitations (which are mostly technical) that need to be addressed before the full potential of the genome database(s) can be realized are emphasized.

Background Schistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking. Methodology/Principal Findings In this study, we used in vitro-grown schistosomula (aged between 2 to 14 days) to investigate drug effects of the three anthelmintics praziquantel, artemether, and oxamniquine. Further, we analyzed the antibody accessibility of two exemplary schistosome antigens SmCD59a and SmKK7, before and after drug treatment. Our results demonstrated that praziquantel applied at a concentration of 1 μM inhibited development of all life stages. Application of 10 μM praziquantel led to dramatic morphological changes of all schistosomula. Artemether at 1 and 10 μM had differential effects depending on whether it was applied to 2-day as compared to 7- and 14-day schistosomula. While 2-day schistosomula were not killed but inhibited from further development, severe morphological damage was seen in 7- and 14-day schistosomula. Oxamniquine (1 and 10 μM) led to severe morphological impairment in all life stages. Analyzing the accessibility of the antigens SmCD59a and SmKK7 before drug treatment showed no antibody binding on living intact schistosomula. However, when schistosomula were treated with anthelmintics, both antigens became exposed on the larvae. Oxamniquine turned out to be most effective in promoting antibody binding to all schistosomula stages. Conclusion This study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack., Author Summary Schistosomiasis is one of the major parasitic diseases in developing countries and still causes 200,000 deaths per year. Mass drug administration programs with praziquantel, the drug of choice against schistosomiasis, are currently undertaken in Sub-Saharan Africa. Praziquantel, although efficient against adult worms, fails to cure early infection. The complex developmental stages of schistosomes and migration through varying host compartments with different local drug concentration are a challenge for drug treatment. After infecting their mammalian host, schistosomula traverse through skin and the vasculature of lung, liver and intestines. During their migration, they develop from larvae to paired adults in approximately 4 to 5 weeks. So far, drug effects have been analyzed on adult worms or freshly transformed schistosomula only. Information about the effects on the larval stages is lacking. We were able to transfer the larval development of the first three weeks into the culture dish. This tool can be used for the analysis of drug effects against schistosomula and for investigation of the accessibility, expression and localization of antigens. Rendering the parasite’s larvae vulnerable to the host’s immune system by increasing antigen presentation is an important aspect of drug activity. We demonstrate on in vitro-cultured Schistosoma mansoni larvae, that SmCD59a and SmKK7, as examples for hidden antigens, become accessible to antibodies following drug treatment.

Background Rhesus macaques are unusual among schistosome hosts, self-curing from an established infection and thereafter manifesting solid immunity against a challenge, an ideal model for vaccine development. Previously, the immunological basis of self-cure was confirmed; surviving worms had ceased feeding but how immunological pressure achieved this was unclear. The schistosome esophagus is not simply a conduit for blood but plays a central role in its processing. Secretions from the anterior and posterior esophageal glands mix with incoming blood causing erythrocyte lysis and tethering and killing of leucocytes. Methodology/Principal Findings We have analysed the self-cure process in rhesus macaques infected with Schistosoma japonicum. Faecal egg output and circulating antigen levels were used to chart the establishment of a mature worm population and its subsequent demise. The physiological stress of surviving females at perfusion was especially evident from their pale, shrunken appearance, while changes in the structure and function of the esophagus were observed in both sexes. In the anterior region electron microscopy revealed that the vesicle secretory process was disrupted, the tips of lining corrugations being swollen by greatly enlarged vesicles and the putative sites of vesicle release obscured by intense deposits of IgG. The lumen of the posterior esophagus in starving worms was occluded by cellular debris and the lining cytoplasmic plates were closely adherent, also potentially preventing secretion. Seven proteins secreted by the posterior gland were identified and IgG responses were detected to some or all of them. Intrinsic rhesus IgG colocalized with secreted SjMEGs 4.1, 8.2, 9, 11 and VAL-7 on cryosections, suggesting they are potential targets for disruption of function. Conclusions/Significance Our data suggest that rhesus macaques self-cure by blocking esophagus function with antibody; the protein products of the glands provide a new class of potential vaccine targets., Author Summary Rhesus macaques can self-cure from a schistosome infection. Antibody is crucial to drive this process and adult worm elimination is preceded by cessation of blood feeding. Recently we have shown that the schistosome esophagus plays a central role in blood processing. We first confirm the self-cure process in rhesus macaques infected with Schistosoma japonicum and provide evidence that the self-cure mechanism involves blocking the worm esophagus function with antibody. In the anterior region, secretion of light vesicles is disrupted hence their contents are not released into the lumen to interact with blood components to fulfil their tasks. The plates in the posterior lining stick together whilst the lumen is occluded, hampering blood processing. Furthermore, rhesus IgG binds strongly to the worm esophageal lumen and co-localizes completely with five esophageal secreted proteins, SjMEGs 4.1, 8.2, 9, 11 and VAL-7. Our results indicate that rhesus macaques eliminate their adult worms by disrupting esophageal function making blood difficult to ingest; feeding stops eventually causing their demise because nutrient uptake across the body surface cannot fully compensate.

Schistosoma haematobium affects more than 100 million people throughout Africa and is the causative agent of urogenital schistosomiasis. The parasite is strongly associated with urothelial cancer in infected individuals and as such is designated a group I carcinogen by the International Agency for Research on Cancer. Using a protein microarray containing schistosome proteins, we sought to identify antigens that were the targets of protective IgG1 immune responses in S. haematobium-exposed individuals that acquire drug-induced resistance (DIR) to schistosomiasis after praziquantel treatment. Numerous antigens with known vaccine potential were identified, including calpain (Smp80), tetraspanins, glutathione-S-transferases and glucose transporters (SGTP1), as well as previously uncharacterized proteins. Reactive IgG1 responses were not elevated in exposed individuals who did not acquire DIR. To complement our human subjects study, we screened for antigen targets of rhesus macaques rendered resistant to Schistosoma japonicum by experimental infection followed by self-cure, and discovered a number of new and known vaccine targets, including major targets recognised by our human subjects. This study has further validated the immunomics-based approach to schistosomiasis vaccine antigen discovery and identified numerous novel potential vaccine antigens.

Study of proteins located at the host–parasite interface in schistosomes might provide clues about the mechanisms utilized by the parasite to escape the host immune system attack. Micro-exon gene (MEG) protein products and venom allergen-like (VAL) proteins have been shown to be present in schistosome secretions or associated with glands, which led to the hypothesis that they are important components in the molecular interaction of the parasite with the host. Phylogenetic and structural analysis of genes and their transcripts in these two classes shows that recent species-specific expansion of gene number for these families occurred separately in three different species of schistosomes. Enrichment of transposable elements in MEG and VAL genes in Schistosoma mansoni provides a credible mechanism for preferential expansion of gene numbers for these families. Analysis of the ratio between synonymous and nonsynonymous substitution rates (dN/dS) in the comparison between schistosome orthologs for the two classes of genes reveals significantly higher values when compared with a set of a control genes coding for secreted proteins, and for proteins previously localized in the tegument. Additional analyses of paralog genes indicate that exposure of the protein to the definitive host immune system is a determining factor leading to the higher than usual dN/dS values in those genes. The observation that two genes encoding S. mansoni vaccine candidate proteins, known to be exposed at the parasite surface, also display similar evolutionary dynamics suggests a broad response of the parasite to evolutionary pressure imposed by the definitive host immune system.

Background The soluble antigen preparation of adult schistosomes (SWAP) has often been used to probe host responses against these blood-dwelling parasites. Despite its long-established use there is limited knowledge about its composition. The information we provide here on the molecular composition of SWAP may contribute as a guide for a rational selection of antigenic targets. Methods Label-free quantitative shotgun proteomics was employed to determine the composition and abundance of SWAP constituents. Briefly, paired adult Schistosoma mansoni worms were sonicated in PBS pH 7.2 and ultracentrifuged for recovery of the soluble supernatant. An aliquot was subjected to trypsin digestion. Resulting peptides were separated under ultra-high performance liquid chromatography and analysed using an orbitrap mass spectrometer. Spectral data were interrogated using SequestHT against an in-house S. mansoni database. Proteins were quantified by recording the mean area under curve obtained for up to three most intense detected peptides. Proteins within the 90th percentile of the total SWAP mass were categorized according to their sub-cellular/tissue location. Results In this work we expanded significantly the list of known SWAP constituents. Through application of stringent criteria, a total of 633 proteins were quantitatively identified. Only 18 proteins account to 50 % of the total SWAP mass as revealed by their cumulative abundance. Among them, none is predicted as a secreted molecule reinforcing the point that SWAP is dominated by cytosolic and cytoskeletal proteins. In contrast, only 3 % of the mass comprised proteins proposed to function at the host-parasite interfaces (tegument and gut), which could conceivably represent vulnerable targets of a protective immune response. Paradoxically, at least 11 SWAP proteins, comprising ~25 % of its mass, have been tested as vaccine candidates. Conclusions Our data suggest that use of SWAP to probe host responses has greatest value for diagnostic purposes or assessing intensity of infection. However, the preparation is of limited utility as an antigen source for investigating host responses to proteins expressed at or secreted from worm-host interfaces. The data also pose the question as to why vaccination with SWAP, containing so many proposed vaccine candidates, has no additive or even synergistic effects on the induction of protection. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-0943-x) contains supplementary material, which is available to authorized users.

Background The schistosome esophagus is divided into anterior and posterior compartments, each surrounded by a dense cluster of gland cell bodies, the source of distinct secretory vesicles discharged into the lumen to initiate the processing of ingested blood. Erythrocytes are lysed in the lumen, leucocytes are tethered and killed and platelets are eliminated. We know little about the proteins secreted from the two glands that mediate these biological processes. Methodology/Principal Findings We have used subtractive RNA-Seq to characterise the complement of genes that are differentially expressed in a head preparation, compared to matched tissues from worm tails. The expression site of representative highlighted genes was then validated using whole munt in situ hybridisation (WISH). Mapping of transcript reads to the S. mansoni genome assembly using Cufflinks identified ~90 genes that were differentially expressed >fourfold in the head preparation; ~50 novel transcripts were also identified by de novo assembly using Trinity. The largest subset (27) of secreted proteins was encoded by microexon genes (MEGs), the most intense focus identified to date. Expression of three (MEGs 12, 16, 17) was confirmed in the anterior gland and five (MEGs 8.1, 9, 11, 15 and 22) in the posterior gland. The other major subset comprised nine lysosomal hydrolases (aspartyl proteases, phospholipases and palmitoyl thioesterase), again localised to the glands. Conclusions A proportion of the MEG-encoded secretory proteins can be classified by their primary structure. We have suggested testable hypotheses about how they might function, in conjunction with the lysosomal hydrolases, to mediate the biological processes that occur in the esophagus lumen. Antibodies bind to the esophageal secretions in both permissive and self-curing hosts, suggesting that the proteins represent a novel panel of untested vaccine candidates. A second major task is to identify which of them can serve as immune targets., Author Summary Schistosomes feed on blood and we have previously shown that its processing begins in the esophagus, which does not act simply as a conduit. It comprises anterior and posterior compartments, each surrounded by glands that secrete proteins into the lumen. Erythrocytes are ruptured as they pass through the compartments and leucocytes are tethered and killed but blood fails to clot. We wanted to identify the proteins secreted from these glands by sequencing the transcriptomes of head and tail preparations to pinpoint those messenger RNAs predominantly or exclusively present only in the heads. We found approximately 50 such proteins, the largest group of 27 being encoded by microexon genes. A second group comprised hydrolytic enzymes that operate at an acid pH. We showed by hybridisation experiments that expression of these genes is indeed localised to either the anterior or the posterior gland. We have suggested that this complex mixture of secreted proteins act together to perform the biological processes that occur in the lumen or, in the case of O-glycosylated membrane proteins, form a protective lining coat. We now want to discover which of them can serve as immune targets in infected animal hosts.

A current or previous schistosome infection might compromise the efficacy of a schistosome vaccine administered to humans. We have therefore investigated the influence of infection on vaccination, using the baboon as the model host and irradiatedSchistosoma mansonicercariae as the vaccine. Protection, determined from worm burdens in test and controls, was not diminished when vaccination was superimposed on a chronic infection, nor was it diminished when it followed a primary infection terminated by chemotherapy. Protection was also assessed indirectly based on fecal egg output and circulating antigen levels, as would be the case in human vaccine trials. In almost all instances, these methods overestimated protection, sometimes with discrepancies of >20%. The overwhelming immune response to egg deposition in infected animals made it difficult to discern a contribution from vaccination. Nevertheless, the well-documented immunomodulation of immune responses that follows egg deposition did not appear to impede the protective mechanisms elicited by vaccination with attenuated cercariae.

Schistosomiasis, caused by parasitic helminths, remains a serious human disease in the tropics. Cercariae of Schistosoma mansoni infect their hosts by direct skin penetration, aided by secretions from acetabular and head glands. Both proteolytic and immunomodulatory properties have been ascribed to the released material, but to date only five isoforms of elastase and one putative anti-inflammatory protein (Sm16) have been cloned. We analyzed secretions from mechanically transformed cercariae by two-dimensional electrophoresis. An average gel image was created and compared with a separation of soluble larval extract, revealing a less complex spot pattern in the secretions with 60% of the spots matched to the larval extract. Subsequent tandem mass spectrometric analysis identified 48 spots from the released material, representing approximately 80% of its normalized volume. Twenty-nine of these are likely to originate in the vesicles, and 18 are likely to originate in the cytosol of the glands (the latter class being present due to holocrine secretion); one is unknown. The vesicular proteins were significantly more enriched than the cytosolic proteins in the released material when compared with the larval extract. A novel metalloproteinase (termed SmPepM8) was the second most abundant constituent after three isoforms of cercarial elastase. In addition, a dipeptidyl peptidase IV (SmDPP IV) was discovered but in much smaller quantity. A new serine protease inhibitor (SmSerp_c) was also prominent. Along with Sm16, four potential immunomodulators were identified, three with similarity to venom allergens (SmSCP_a, _b, and _c) and one with homology to the potassium channel blockers in scorpion venom (SmKK7). Interrogation of the expressed sequence tag database found transcripts encoding the majority of vesicular proteins present solely in the intramolluscan stages of the life cycle. Distinct patterns of radiolabel incorporation suggested three separate origins for the vesicular proteins. All the novel constituents merit investigation as vaccine candidates, and the potential immunomodulators merit investigation as therapeutic agents.

The blood fluke Schistosoma mansoni can live for years in the hepatic portal system of its human host and so must possess very effective mechanisms of immune evasion. The key to understanding how these operate lies in defining the molecular organisation of the exposed parasite surface. The adult worm is covered by a syncytial tegument, bounded externally by a plasma membrane and overlain by a laminate secretion, the membranocalyx. In order to determine the protein composition of this surface, the membranes were detached using a freeze/thaw technique and enriched by sucrose density gradient centrifugation. The resulting preparation was sequentially extracted with three reagents of increasing solubilising power. The extracts were separated by 2-DE and their protein constituents were identified by MS/MS, yielding predominantly cytosolic, cytoskeletal and membrane-associated proteins, respectively. After extraction, the final pellet containing membrane-spanning proteins was processed by liquid chromatographic techniques before MS. Transporters for sugars, amino acids, ions and other solutes were found together with membrane enzymes and proteins concerned with membrane structure. The proteins identified were categorised by their function and putative location on the basis of their homology with annotated proteins in other organisms.

The lung schistosomulum of Schistosoma mansoni is a validated target of protective immunity elicited in vaccinated mice. To identify genes expressed at this stage we constructed a microarray, representing 3088 contigs and singlets, with cDNA derived from in vitro cultured larvae and used it to screen RNA from seven life-cycle stages. Clustering of genes by expression profile across the life cycle revealed a number of membrane, membrane-associated and secreted proteins up-regulated at the lung stage, that may represent potential immune targets. Two promising secreted molecules have homology to antigens with vaccine and/or immunomodulatory potential in other helminths.

Background: The esophagus of blood-feeding schistosomes has been largely neglected although its posterior portion was designated as a gland decades ago. However, we recently showed it plays a pivotal role in blood processing. It is clearly demarcated into anterior and posterior compartments, both surrounded by a mass of cell bodies. Feeding movies revealed that erythrocytes accumulate in the anterior compartment before entering the posterior, indicating that a distinct process is executed there. We therefore investigated ultrastructural aspects and possible functions of the anterior region. Methods: The heads of adult Schistosoma japonicum were detached and prepared for both transmission and scanning electron microscopy to define the detailed ultrastructure of the anterior esophagus. Cryosections of heads were also prepared for immunocytochemistry and confocal microscopy to define the pattern of intrinsic host antibody binding in the anterior esophageal lining. Results: The anterior syncytial lining of the esophagus is highly extended by long, thin corrugations of cytoplasm projecting towards the lumen. Strikingly in the male worm, the tips of the corrugations are further expanded by numerous threads of cytoplasm, producing a spaghetti-like appearance in the central lumen. Flattened, pitted cytoplasmic plates are interspersed in the tangled mass of threads. Abundant, morphologically distinct light vesicles of varied size and contents are manufactured in the cell bodies, from where they traffic through cytoplasmic connections to the corrugations and out to the tips. Clusters of vesicles accumulate in expanded tips in males, together with occasional mitochondria whilst females have more mitochondria but fewer vesicles. The membranous contents of light vesicles are secreted mainly from the tips, but also from the sides of the corrugations. They coat the surfaces and then form organised self-adherent membrane figures when shed into the lumen. Host antibody binds strongly in a characteristic pattern to the anterior esophageal lining indicating that the secretions are highly immunogenic. Conclusions: We suggest that the anterior esophageal region is an independent secretory organ. The contents of light vesicles are released into the esophageal lumen via the tips of corrugation to interact with incoming blood. Our immediate task is to establish their composition and role in blood processing.

Schistosomiasis is one of the most prevalent parasitic diseases, affecting millions of people in developing countries. Amongst the human-infective species, Schistosoma mansoni is also the most commonly used in the laboratory and here we present the systematic improvement of its draft genome. We used Sanger capillary and deep-coverage Illumina sequencing from clonal worms to upgrade the highly fragmented draft 380 Mb genome to one with only 885 scaffolds and more than 81% of the bases organised into chromosomes. We have also used transcriptome sequencing (RNA-seq) from four time points in the parasite's life cycle to refine gene predictions and profile their expression. More than 45% of predicted genes have been extensively modified and the total number has been reduced from 11,807 to 10,852. Using the new version of the genome, we identified trans-splicing events occurring in at least 11% of genes and identified clear cases where it is used to resolve polycistronic transcripts. We have produced a high-resolution map of temporal changes in expression for 9,535 genes, covering an unprecedented dynamic range for this organism. All of these data have been consolidated into a searchable format within the GeneDB (www.genedb.org) and SchistoDB (www.schistodb.net) databases. With further transcriptional profiling and genome sequencing increasingly accessible, the upgraded genome will form a fundamental dataset to underpin further advances in schistosome research., Author Summary Schistosomiasis is a disease caused by parasitic blood flukes of the genus Schistosoma. Human-infective species are prevalent in developing countries, where they represent a major disease burden as well as an impediment to socioeconomic development. In addition to its clinical relevance, Schistosoma mansoni is the species most widely used for laboratory experimentation. In 2009, the first draft of the S. mansoni and S. japonicum genomes were published. Both genome sequences represented a great step forward for schistosome research, but their highly fragmented nature compromised the quality of potential downstream analyses. In this study, we have substantially improved both the genome and the transcriptome resources for S. mansoni. We collated existing data and added deep DNA sequence data from clonal worms and RNA sequence data from four key time points in the life cycle of the parasite. We were able to identify transcribed regions to single-base resolution and have profiled gene expression from the free-living larvae to the early human parasitic stage. We uncovered extensive use of single transcripts from multiple genes, which the organism subsequently resolves by trans-splicing. All data from this study comprise a major new release of the genome, which is publicly and easily accessible.

Progress on schistosome research has, until recently, suffered from limited genomic information. This situation changed dramatically in October 2003 with the simultaneous publication of the Schistosoma mansoni and Schistosome japonicum transcriptomes. A total of 45 000 expressed-sequence tags was obtained for S. japonicum, derived from adult worms and eggs. For S. mansoni, a total of ∼125 000 ESTs was analyzed, derived from six different life cycle stages, and estimated to represent 92% of the complement of >14 000 genes. The wealth of information generated is essential for annotation of the now-sequenced S. mansoni genome. It has permitted a list of potential drug targets and vaccine candidates to be drawn up, and has set in train proteomic and functional genomic projects to exploit the data, and ultimately lead to improved control strategies.

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.

The process of skin invasion by Schistosoma cercariae was reviewed in a recent Trends Research Update, accompanied by a computer animation. Some aspects of that article were misleading and perpetuated misconceptions about parasite migration through the skin that should by now have been dispelled. This article sets out a different interpretation of events, taking account of the extensive data on migration and larval structural changes that have been documented for Schistosoma mansoni over the past 20 years.

SUMMARYThe potential of various quantitative lateral flow (LF) based assays utilizing up-converting phosphor (UCP) reporters for the diagnosis of schistosomiasis is reviewed including recent developments. Active infections are demonstrated by screening for the presence of regurgitated worm antigens (genus specific polysaccharides), whereas anti-Schistosomaantibodies may indicate ongoing as well as past infections. The circulating anodic antigen (CAA) in serum or urine (and potentially also saliva) is identified as the marker that may allow detection of single-worm infections. Quantitation of antigen levels is a reliable method to study effects of drug administration, worm burden and anti-fecundity mechanisms. Moreover, the ratio of CAA and circulating cathodic antigen (CCA) is postulated to facilitate identification of eitherSchistosoma mansoniorSchistosoma haematobiuminfections. The UCP-LF assays allow simultaneous detection of multiple targets on a single strip, a valuable feature for antibody detection assays. Although antibody detection in endemic regions is not a useful tool to diagnose active infections, it gains potential when the ratio of different classes of antibody specific for the parasite/disease can be determined. The UCP-LF antibody assay format allows this type of multiplexing, including testing a linear array of up to 20 different targets. Multiple test spots would allow detection of specific antibodies, e.g. against differentSchistosomaspecies or other pathogens as soil-transmitted helminths. Concluding, the different UCP-LF based assays for diagnosis of schistosomiasis provide a collection of tests with relatively low complexity and high sensitivity, covering the full range of diagnostics needed in control programmes for mapping, screening and monitoring.