A Schistosoma protein, Sh-TOR, is a novel inhibitor of complement which binds human C2

Human complement regulatory (also called inhibitory) proteins control misdirected attack of complement against autologous cells. Trypanosome and schistosome parasites which survive in the host vascular system also possess regulators of human complement. We have shown Sh-TOR, a protein with three predicted transmembrane domains, located on the Schistosoma parasite surface, to be a novel complement regulatory receptor. The N-terminal extracellular domain, Sh-TOR-ed1, binds the complement protein C2 from human serum and specifically interacts with the C2a fragment. As a result Sh-TOR-ed1 pre-incubated with C2 inhibits classical pathway (CP)-mediated haemolysis of sheep erythrocytes in a dose-dependent manner. In CP-mediated complement activation, C2 normally binds to C4b to form the CP C3 convertase and Sh-TOR-ed1 has short regions of sequence identity with a segment of human C4b. We propose the more appropriate name for TOR of CRIT (complement C2 receptor inhibitory trispanning).     

Human neutrophils endocytose multivalent ligands from the surface of schistosomula of Schistosoma mansoni before membrane fusion

Human buffy coat cells adhering to schistosomula of Schistosoma mansoni that were preincubated in fluorochrome-conjugated concanavalin A (Con A), wheat germ agglutinin, lentil lectin, or purified IgG from a hyperimmunized rabbit, were examined by fluorescence and transmission electron microscopy and by freeze-fracture. All four fluorochrome- conjugated multivalent ligands were homogeneously distributed on the parasite surface after preincubation. Within 1-3 h after the addition of cells, large areas of nonfluorescence, 10-20 micrometer in diameter, were seen on the parasite surface. In addition, the fluorochromes were observed in granules within the cells. Electron microscope autoradiography of worms preincubated with 125I-Con A showed silver grains evenly distributed over the tegumental membrane. After the addition of cells, grains were seen over phagolysosomes in the cytoplasm of neutrophils adhering to the parasites. In addition, no grains were present over large areas of the tegumental membrane, which still retained its normal architecture, or over fusions between the neutrophil plasma membrane and the outer tegumental membrane. Rabbit IgG formed an electron-dense layer on the tegumental membrane which was endocytosed by neutrophils. Both neutrophils and eosinophils fused with the parasite in areas containing no electron-dense material on the surface. It is concluded that human neutrophils will endocytose a variety of multivalent ligands from the surface of schistosomula, which probably accounts for the failure of neutrophils to kill the parasite and acts to clear the parasite surface of both antigen and antibody. Presumably, the components of the parasite surface which have originally bound the ligands are also endocytosed since surface components labeled by galactose oxidase and NaB3H4 are taken into cells when examined by light microscope autoradiography. Finally, membrane fusion occurs in areas devoid of multivalent glands, which suggests that these ligands serve to bring the cells and parasites close together, but the actual fusigens probably reside in the lipids in the outer tegumental membrane.     

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.     

Mapping of the N terminus of the Schistosoma mansoni tegumental antigen Sm15 to its predicted precursor protein

Sm15 is a major Schistosoma mansoni 15 kDa tegumental antigen, resulting from the proteolytic processing of a larger precursor. The amino terminus of Sm15 was identified by direct amino acid sequencing, and the antigen was tentatively mapped to the segment spanning amino acids 362-497 of the precursor. This will allow subsequent studies to elucidate the possible immunological role of proteolytic processing in schistosomiasis.     

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling.     

Schistosoma mansoni: identification, characterization, and purification of the spine glycoprotein by monoclonal antibody

A tegumental surface membrane antigen of Schistosoma mansoni has been identified by use of a monoclonal antibody. The binding of ¹²⁵I-labeled monoclonal antibody showed that proteins sharing antigenic determinants recognized by this monoclonal antibody were present in cercariae and worms of both sexes, but were absent from schistosome egg extract. The protein molecules expressing these antigenic determinants differed in molecular weight: 120,000 in cercaria and 170,000 in male and female worms. The cercarial glycoprotein immunoprecipitated with the monoclonal antibody was also immunoprecipited by sera of infected humans, as shown by two-dimensional gel electrophoresis and tryptic peptide mapping. The location of the glycoprotein identified by the monoclonal antibody was restricted to the spines of the schistosomular surface, the tubercle-associated spines of the male worm, and the dorsal spines of the female worm. The spine glycoprotein was readily purified by immunoaffinity chromatography. These findings are discussed in relation to parasite development and the relevance of this antibody for serodiagnosis and immunoprophylaxis.     

An immunogenic Mr 23,000 integral membrane protein of Schistosoma mansoni worms that closely resembles a human tumor-associated antigen

A Mr 23,000 Ag of the human trematode parasite, Schistosoma mansoni, has been identified by immunoscreening an adult worm cDNA library with antibody affinity purified on the Mr 23,000 to 25,000 integral membrane protein fraction of the parasite. This Ag is immunogenic in infected humans as well as in rabbits exposed to S. mansoni. The protein sequence of the Ag as deduced from cloned DNA sequences is 218 amino acids long and contains four putative transmembrane regions. Of particular significance, the Ag is strikingly similar, with respect to both amino acid sequence (36% identity) and putative domain structure to ME491, a human stage-specific melanoma-associated Ag.     

Schistosoma mansoni: a complement-dependent receptor on adult male parasites

The dorsal surface of adult male Schistosoma mansoni exhibits an affinity for Salmonella typhi when these bacteria are preincubated in normal serum from mice, guinea pigs, or humans. The complement (C) system was shown to be responsible for the bacterial binding. Bacteria not preincubated with normal serum or preincubated in normal serum which had been treated with ethylenediamine tetraacetic acid (EDTA), or cobra venom factor (CoF), or heated at 56 C for 60 min did not bind to the parasite's surface. Further experiments utilizing ethylene glycol tetraacetic acid (EGTA) plus Mg²⁺, heat inactivation at 50 C for 30 min, and zymosan treatment of the serum indicated the C fixation and deposition on the bacteria occurs via the alternative C pathway. These observations indicate the presence of a complement-dependent receptor on the dorsal tegumental surface of the adult male parasite.     

Schistosoma mansoni: surface membrane stability in vitro and in vivo

The human complement component C3b is known to bind in vitro to the surfaces of all developmental stages of schistosomes as a consequence of complement activation by the alternative pathway. C3b bound to Schistosoma mansoni parasites has now been used in combination with fluorescent labeled antibodies against C3b to label the surfaces of living schistosomes. Binding of complement components and labeled antibodies to adult schistosomes rendered their surface membrane homogeneously fluorescent. At the ultrastructural level, the label was seen as a dense deposit lying on the tegumental membrane. Surface damage was not observed in labeled adults by electron microscopy. Fluorescent schistosomes were cultured in vitro for periods of up to 2 weeks, during which time the parasites remained fully viable and their surface membrane was still fluorescent. The electron dense deposit persisted, and tegumental damage at the electron microscope level was minimal or absent. Consequently, adult schistosomes would seem able to survive in vitro in the absence of rapid and general turnover of their surface membrane. Loss of fluorescence was observed consistently only at the anterior end of the parasite, including the suckers, a finding which indicates that membrane turnover may occur at different rates on different parts of the body. Fluorescent 3-week-old juveniles and 6-day-old lung stage parasites were cultured under the same conditions with similar results: they remained viable and fluorescent for at least 2 weeks. Results with skin schistosomula were different in the sense that many worms died during culture, and those which survived lost large parts of their fluorescent surface. A few of the surviving and fluorescent schistosomula developed the elongate shape typical of lung stage parasites. Fluorescent viable skin schistosomula were injected intravenously into mice and subsequently recovered from the lungs after varying periods. Fluorescence was lost in a patchy way within a few minutes from some individuals and within several hours from most of the worms. These data permit the following conclusions: C3b is a suitable tracer for membrane renewal in all developmental stages of schistosomes. Very slow membrane renewal in vitro and very rapid renewal in vivo are both compatible with parasite survival.     

Schistosoma mansoni: the dicer gene and its expression

RNA interference (RNAi) is a gene silencing mechanism that plays an important role in regulating gene expression in many eukaryotes and has become a valuable molecular tool for analyzing gene function. Multi-domain nucleases called Dicer proteins play pivotal roles in RNAi. In this paper, we characterize the structure and expression of the Dicer gene from the platyhelminth parasite Schistosoma mansoni. The gene (SmDicer) is over 54kb long and comprises 30 exons that potentially encode a 2641 amino acid protein. This is the largest Dicer protein yet described. SmDicer contains all domains that are characteristic of metazoan dicers including an amino terminal helicase domain, DUF283, a PAZ domain, two RNAse III domains and an RNA binding domain. An examination of the available S. mansoni genome sequence suggests that the Dicer gene described here is the only Dicer gene in the parasite genome. SmDicer is expressed throughout schistosome development suggesting that RNAi technologies might be employed in deciphering gene function in all life stages of this parasite.     

Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome

Schistosoma mansoni is a parasitic worm that lives in the blood vessels of its host. We mapped the S. mansoni tegumental outer-surface structure proteome by 1D SDS-PAGE and LC-MS/MS and an EST-database from the ongoing genome-sequencing project. We identified 740 proteins of which 43 were tegument-specific. Many of these proteins show no homology to any nonschistosomal protein, demonstrating that the schistosomal outer-surface comprises specific and unique proteins, likely to be critical for parasite survival.     

Partial and complete detachment of neutrophils and eosinophils from schistosomula: evidence for the establishment of continuity between a fused and normal parasite membrane

Neutrophils and eosinophils adhering to the surface of schistosomula of Schistosoma mansoni have been partially or completely detached with hypertonic sucrose or by pipetting. The sucrose-treated neutrophils are attached only in areas where there are pentalaminar fusions between the neutrophil and tegumental membranes, suggesting that these fusions attach the cells to the parasites. Pipetting breaks many of the attached cells. In thin section, the tegumental membrane underlying these cells is seen to be pentalaminar. By freeze-fracture techniques, modified attachment areas are found. The edge zone often appears as a single strand of intramembrane particles (IMPs) on the P2 face and as a groove on the E2 face. The edge zone may also have large discontinuities, in which case it no longer separates membrane faces of unequal IMP density from one another. In addition, the IMPs on the IMP- rich areas become aggregated and surrounded by craters in the membrane. These experiments suggest that the fusions may be the mechanism by which the parasite acquires some host membrane components on its surface. On the other hand, eosinophil plasma membranes are seen adhering to a layer of electron-dense material on the parasite after the cells have been disrupted by pipetting. This suggests that eosinophils adhere to the parasite surface through their discharged granule material and not by membrane fusions.     

Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized within the tegument of fixed worms on the cytoplasmic leaflet of both the double surface membrane and the basement membrane; this reaction was inhibited by 1 microM chlorpromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosomes are discussed.     

Schistosoma mansoni: Surface Membrane Isolation with Lectin-Coated Beads

Lectins from Lens culinaris and Arachis hypogaea immobilized on polyacrylamide beads were used for selective isolation of glycosylated surface membrane domains of adult Schistosoma mansoni worms, and the method was compared with the membrane isolation procedure developed with polycationic (Affi-Gel) beads. The lentil lectin proved to be suitable for interaction with surface membrane components: an increment in the specific activities of tegumental phosphohydrolases was observed in the bound fraction with respect to that observed in a total worm homogenate. A characteristic polypeptide pattern on gel electrophoresis was also seen, more restricted than that obtained with the bound Affi-Gel fraction. Immobilized peanut lectin was not successful as a method for isolating membrane material from the tegument of adult worms. Solubilization and dissociation of the lentil lectin-bound enzyme markers was achieved after addition of detergent and competing sugars. Glycosylation of the solubilized enzymes was further confirmed by affinity chromatography with fresh lentil lectin-coated beads. These results, together with histochemical evidences, suggest that the active sites of some of these enzymes are locted within or close to the cytoplasmic leaflet of the surface tegumental membranes, and allow us to propose a model for the double surface membrane complex where some proteins may be crossing the two bilayers.     

T and B cell epitope mapping of SM23, an integral membrane protein of Schistosoma mansoni.

SM23 is an integral membrane protein of the blood-vessel dwelling parasitic worm Schistosoma mansoni. This protein has been detected with antibodies in all stages of the parasite found in the human host, notably the lung stage, and therefore is of interest as a vaccine candidate. In addition SM23 has been shown to be a member of a proposed new superfamily of membrane proteins whose structures do not conform to the previously known classifications. To date there are 13 members including ME491 (CD63, Pltgp40), CD9 (p23), TAPA-1, CD37, CD53, MRC OX-44, CO-029, MRP-1, L6, the gene product of TI-1, the target of mAb AD-1, SM23, and SJ23 (the Schistosoma japonicum homologue). Most of these molecules except for those in the two blood vessel-dwelling parasites are found in membranes of hemopoietic and/or malignant cells and all have unknown function. In this study we used recombinantly expressed full-length and partial molecules as well as synthesized peptides to map T cell and B cell epitopes of SM23. The two predicted external hydrophilic domains were found to be highly immunogenic and contained several B cell epitopes. There were at least four T cell epitopes in the large hydrophilic domain. One segment of 23 amino acids contained both a T cell and B cell epitope as well as the putative glycosylation site. This particular segment was recognized by immune sera and cells of every mouse strain tested. The elucidation of these epitopes demonstrates the immunogenic nature of this molecule and raises questions as to the role of SM23 in the host/parasite relationship.     

Exposed proteins of the Schistosoma japonicum tegument

The ability of the mammalian blood fluke Schistosoma japonicum to survive in the inhospitable environment of the mammalian bloodstream can be attributed, at least in part, to its host-exposed outer surface, called the tegument. The tegument is a dynamic organ and is involved in nutrition, immune evasion and modulation, excretion, osmoregulation and signal transduction. Given its importance for parasite survival, proteins exposed to the host at the surface of the tegument are ideal targets for the development of vaccines and drugs. By biotinylating live adult worms and using a combination of OFFGEL electrophoresis and tandem mass spectrometry 54 proteins were identified as putatively host-exposed in S. japonicum. These included glucose transport proteins, an amino permease, a leucine aminopeptidase and a range of transporters, heat shock proteins and novel immune-active proteins. Members of the tetraspanin protein family and a homologue of Sm 29, a tegument membrane protein from Schistosoma mansoni, both effective vaccine antigens in S. mansoni, were also identified. The fate of labelled surface proteins was monitored over time using electron microscopy and revealed that biotinylated proteins were rapidly internalised from the surface of the tegument and trafficked into the cytoplasmic bridges that connect the distal cytoplasm of the tegument to the underlying cell bodies. The results reported herein dramatically increase the number of S. japonicum proteins known to be exposed to the host and, hence, those of interest as therapeutic targets. The ability of the parasite to rapidly internalise proteins at its surface has implications for the development of vaccines and may explain how these parasites are able to avoid the host immune system for long periods of time.     

Characterisation of a family of Schistosoma japonicum proteins related to dynein light chains.

Dynein light chains (DLC) are components of dynein, an enzyme complex involved in various aspects of microtubule-based motility. We report here the molecular cloning and sequencing of cDNAs encoding a family of DLC-like polypeptides (SjcDLC1-5) from the human bloodfluke Schistosoma japonicum with open reading frames of 87-104 amino acids and deduced molecular masses ranging from 10.5 to 12.3 kDa. Two-dimensional Western blot analysis confirmed the presence of several S. japonicum DLC isoforms with differing pI values and molecular sizes. We also describe the molecular characterisation, genomic organisation and expression of clone SjcDLC1, and the immunological characterisation and localisation of its encoded protein. Northern blot analysis of adult worm RNA indicated SjcDLC1 is encoded by a single message of approximately 650 bp and Southern analysis suggested one SjcDLC1 gene exists in the S. japonicum genome. Immunolocalisation studies demonstrated that the SjcDLC1 protein is present in the tegument of the adult and cercarial stages of S. japonicum. SjcDLC1 and the other SjcDLC may function in the transport of specialised organelles, comprising membranous and discoid bodies, through the tegument to the schistosome-unique heptalaminate tegumental membrane at the external surface of the adult worm. As a consequence, they may provide novel targets for anti-schistosome vaccine and/or drug development.     

Acetylcholinesterase of Schistosoma mansoni--functional correlates. Contributed in honor of Professor Hans Neurath's 90th birthday

Acetylcholinesterase (AChE) is an enzyme broadly distributed in many species, including parasites. It occurs in multiple molecular forms that differ in their quaternary structure and mode of anchoring to the cell surface. This review summarizes biochemical and immunological investigations carried out in our laboratories on AChE of the helmint, Schistosoma mansoni. AChE appears in S. mansoni in two principal molecular forms, both globular, with sedimentation coefficients of approximately 6.5 and 8 S. On the basis of their substrate specificity and sensitivity to inhibitors, both are "true" acetylcholinesterases. Approximately half of the AChE activity of S. mansoni is located on the outer surface of the parasite, attached to the tegumental membrane via a covalently attached glycosylphosphatidylinositol anchor. The remainder is located within the parasite, mainly associated with muscle tissue. Whereas the internal enzyme is most likely involved in termination of neurotransmission at cholinergic synapses, the role of the surface enzyme remains to be established; there are, however, indications that it is involved in signal transduction. The two forms of AChE differ in their heparin-binding properties, only the internal 8 S form of the AChE being retained on a heparin column. The two forms differ also in their immunological specificity, since they are selectively recognized by different monoclonal antibodies. Polyclonal antibodies raised against S. mansoni AChE purified by affinity chromatography are specific for the parasite AChE, reacting with both molecular forms, but do not recognize AChE from other species. They interact with the surface-localized enzyme on the intact organism, and produce almost total complement-dependent killing of the parasite. S. mansoni AChE is thus demonstrated to be a functional protein, involved in multifaceted activities, which can serve as a suitable candidate for diagnostic purposes, vaccine development, and drug design.