Surface antigens on cercariae,schistosomula and adult worms of Schistosoma mansoni

Shah J. and Ramasamy R. 1982. Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni. International Journal for Parasitology12: 451–461. The surface protein antigens of Schistosoma mansoni were radiolabelled by lactoperoxidase catalysed I¹²⁵-iodination and analysed by immune-precipitation and polyacrylamide gel electrophoresis. The results showed that regularly labelled surface antigens of mol. wts >150,000, 78,000, 45,000 and 22,000 were present on adult worms. Common surface antigens were observed on the cercariae, schistosomula and adult worms. It is suggested that surface antigens released from living adult worms can sensitise a host to react against the invading schistosomula of a secondary infection. However, the failure to vaccinate mice using material containing adult worm surface antigens suggests that the induction of protective immunity is a complex phenomenon.     

Schistosoma mansoni: Demonstration of two circulating antigens in infected hamsters

In this study the presence of two circulating schistosome derived antigens, probably both polysaccharides, was demonstrated in hamsters heavily infected with Schistosoma mansoni. One antigen was an anodic, heat-stable, high molecular weight substance; it was demonstrated in serum, adult worm antigen and in the excretory and secretory products of adult worms. The antigen was demonstrated in the epithelial cells of the schistosome gut. A second antigen, cathodic, heat-stable and a low-molecular weight substance (MW < 30,000), was demonstrated in hamster serum, hamster urine, adult worm antigen, and in the excretory and secretory products of adult worms. Two additional schistosome derived antigens, both heat-labile, were demonstrated in hamster urine.     

Intraspecific Epitopic Variation in a Carbohydrate Antigen Exposed on the Surface of Trichostrongylus colubriformis Infective L3 Larvae

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.     

Schistosoma mansoni: histological localization of gelatinase in the preacetabular glands of cercariae

Proteolytic activity was demonstrated in secretions from the preacetabular glands of cercariae of Schistosoma mansoni. Thin gelatin film substrates were lysed by living cercariae stimulated to penetrate by application on the films of skin surface lipid. Lysis was directly related to number of cercariae, time, and temperature of incubation and pH of the medium. Gelatinase activity in unfixed frozen sections of cercariae incubated on the gelatin films was in the preacetabular glands which are the source of the secretion emptied into skin during penetration. Protease activity, therefore, appears to be related to penetration. The schistosome larvae which made the penetration attempt satisfied the accepted criteria for schistosomules, and therefore appeared to have transformed into schistosomules even though they did not successfully penetrate anything.     

Nicarbazin in schistosome infections: I. Antibody formation in mice and hamsters infected with Schistosoma mansoni.

The immunoprecipitin response of mice infected with Schistosoma mansoni and treated with nicarbazin, an egg suppressive agent, is significantly lower than in untreated-infected mice. The precipitin response to cercarial extract is virtually abolished in treated mice indicating common antigenic determinants with eggs of the same species. Haemagglutinins to adult worms are also significantly diminished in treated mice. Finally, circumoval precipitins are absent in treated mice when the drug is given continuously to infected mice in order to prevent egg laying by the female adult parasite. The results suggest that a significant portion of the precipitating antibody produced in schistosome infections reactive with cercariae and adult worms, as well as eggs, is probably a secondary antibody response due to common antigenic determinants found in eggs.     

Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep

Although gastrointestinal nematode parasites are a major human and veterinary health problem, little is known about how the host is sometimes able to mount an effective immune rejection response. In previous work, we identified a carbohydrate larval surface antigen (CarLA) as the target of mucosal antibodies that can elicit rejection of Trichostrongylus colubriformis L3s in sheep. Here we characterise the natural mucosal antibody responses to L3s from three major strongylid gastrointestinal parasites of sheep, Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. The mucosal antibody repertoire of naturally field-immune sheep was displayed on bacteriophage as single-chain antibodies (scFvs) and phage were selected for the ability to bind to the surface of living L3s of the three nematode species. All nematode-binding scFvs were found to recognize one of three different antigen classes that are each found in the three strongylid species. These three antigen classes appear to represent all of the major antigens recognized on Western blots by pooled mucosal antibodies from field-immune sheep. One of the antigen classes is a heterogeneous, high molecular weight molecule that is protease-sensitive. The scFvs recognizing this surface antigen also recognize a similar antigen in all strongylids tested. A second antigen class is a protease-insensitive, low molecular weight antigen found only in sheaths and scFvs recognizing this antigen cross-react with a similar molecule found in all strongylids tested. The third surface antigen class is CarLA and all of the anti-CarLA scFvs obtained from the field-immune sheep repertoire were specific to L3s of only one species and often recognized only a subset of the worms. Thus three different L3-stage surface antigens, two that lack a protein component, dominate the natural mucosal antibody response to L3-stage gastrointestinal strongylid nematodes in sheep.     

Schistosoma mansoni: Complement activation by antigenic preparations

The ability of antigens prepared from adult worms and eggs of Schistosoma mansoni to activate complement in vitro in normal, human serum in the absence of specific antibodies was investigated. It was demonstrated that whole viable eggs activated the complement system; this was shown to be effected by egg antigens released into the medium. Egg-hatching fluid induced a high degree of complement consumption, whereas purified egg shells gave almost no complement consumption. The complement-activating antigens of the eggs are possibly of polysaccharide nature as indicated by an almost complete complement activation by trichloroacetic acid-soluble egg antigens. No detectable complement consumption occurred upon incubation of living adult worms, but antigens extracted from adult worms did give complement consumption. Circulating cathodic antigens and excretory and secretory antigens proved to be quite capable of inducing complement activation; tegumental antigens gave lower, but still significant levels of complement consumption.     

Innate immunogenicity and in vitro protective potential of Schistosoma mansoni lung schistosomula excretory–secretory candidate vaccine antigens

Excretory–secretory products (ESP) of Schistosoma mansoni developing larvae are ideal potential vaccines as such molecules may readily induce host primary immune responses, and local memory immune response effectors that would target, surround, and pursue the larvae while negotiating the lung blood capillaries. We herein characterized the cytokines response ESP, e.g., SG3PDH, 14-3-3-like protein, TPX, and calpain induce in the natural context of infection, and defined the global cytokine profile conducive to effective schistosome larvae killing. Accordingly, spleen cells (SC) taken from naïve, and 7-, or 9-day S. mansoni-infected mice were stimulated in vitro with the selected ESP, in a recombinant or multiple antigen peptide (MAP) form, and examined for production of T helper type (Th) 1, Th2, and Th17 cytokines, and the ability to mediate in vitro attrition of lung-stage schistosomula. The study indicated that larval ESP principally elicit Th1 and Th17 type cytokines. Recombinant SG3PDH was the only test ESP to additionally activate SC from S. mansoni-infected BALB/c mice to release higher IL-4 levels than unstimulated SC and mediate significant (P < 0.0001) in vitro attrition of lung-stage larvae. Thus, our data suggested that a balance between Th1, Th17, and Th2 cytokines is required for effective schistosome larval elimination.     

Developmental regulation of protein kinase A expression and activity in Schistosoma mansoni

cAMP-dependent protein kinases (PKAs) are the main transducers of cAMP signalling in eukaryotic cells. Recently we reported the identification and characterisation of a PKA catalytic subunit (SmPKA-C) in Schistosoma mansoni that is required for adult schistosome viability in vitro. To gain further insights into the role of SmPKA-C in biological processes during the schistosome life cycle, we undertook a quantitative analysis of SmPKA-C mRNA expression in different life cycle stages. Our data shows that SmPKA-C mRNA expression is developmentally regulated, with the highest levels of expression in cercariae and adult female worms. To evaluate the biological role of SmPKA-C in these developmental stages, cercariae and adult worms were treated with various concentrations of PKA inhibitors. Treatment of cercariae with H-89 or PKI 14-22 amide resulted in loss of viability suggesting that, as in adults, PKA is an essential enzyme activity in this infectious larval stage. In adult worms, in vitro exposure to sub-lethal concentrations of H-89 or PKI 14-22 amide resulted in inhibition of egg production in a dose-dependent manner. Furthermore, using a murine model of schistosome infection where S. mansoni fecundity is impaired, we show that reduced rates of egg production in vivo correlate with significant reductions in SmPKA-C mRNA expression and PKA activity. Finally, restoration of parasite egg production in vivo also resulted in normalisation of SmPKA-C mRNA expression and PKA activity. Taken together, our data suggest that PKA signalling is required for cercarial viability and may play a specific role in the reproductive activity of adult worms.     

Lung-stage expression of a major schistosome surface antigen

The topographical expression of a glycoprotein of 180,000 molecular weight on the surface of lung-stage Schistosoma mansoni schistosomula was determined by immunofluorescence microscopy using a monoclonal antibody. Postfixation treatment with graded ethanols enhanced specific immunofluorescent staining of adult worms, and was required for detection of the antigen on the surfaces of lung-stage schistosomula. The epitope recognized by the monoclonal antibody was also present on the surfaces of adult Schistosoma haematobium, but not on those of Schistosoma japonicum.     

Schistosoma mansoni: Tegumental damage as a consequence of lectin binding

Adult worms of Schistosoma mansoni exhibit gross tegumental damage following incubation in concanavalin A or Ricinus communis agglutinin. However, incubation with wheat germ agglutinin induces only minimal surface damage, while soybean agglutinin has no damaging effect upon the worms. Damage induced by Ricinus communis agglutinin or concanavalin A may be prevented by the addition of the appropriate competing sugar. In contrast, incubation of 3-hr artificially transformed schistosomula in concanavalin A and other lectins does not produce any disruption of the tegument. These results indicate that the surface membrane of the adult schistosome is readily disrupted by ligand binding and appears to be particularly sensitive and fragile. The membrane of the schistosomulum, however, is more resistant to the effects of lectin binding. Adult worms incubated in culture medium alone (ELAC or RPMI 1640) show background changes which seem to be related to the tonicity of the medium. Such results advocate that preliminary assessment of schistosome integrity be carried out prior to any experimental procedures which preclude the addition of serum to the basic incubation medium. Schistosomula do not exhibit comparable sensitivity.     

Schistosoma mansoni U6 gene promoter-driven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules

RNA interference (RNAi) mediated by short hairpin-RNA (shRNA) expressing plasmids can induce specific and long-term knockdown of specific mRNAs in eukaryotic cells. To develop a vector-based RNAi model for Schistosoma mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni. A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 h following transduction with plasmid pXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In a similar fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared with controls. The findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes. Given that the U6 gene promoter drove expression of shRNA from an episome, the findings also indicate the potential of this putative RNA polymerase III dependent promoter as a component regulatory element in vector-based RNAi for functional genomics of schistosomes.     

The anticancer drug imatinib induces autophagy in Schistosoma mansoni

Schistosomiasis, caused by schistosome parasites, is a neglected tropical disease affecting humans and animals. There is no vaccine available yet, and fear of upcoming resistance against the only widely used drug, praziquantel, is omnipresent. Previously, we showed that imatinib (Gleevec), an anticancer drug, affected schistosome physiology and caused the death of adult Schistosoma mansoni in vitro. Here, we present the first known evidence that one effect of imatinib is the induction of autophagy in S. mansoni. Furthermore, worms co-treated with imatinib and bafilomycin A1, a late-phase autophagy inhibitor, reversed imatinib-induced autophagy and its antischistosomal effects as revealed by phenotypic and molecular analyses.     

Schistosoma mansoni: immediate hypersensitivity to adult antigens in the rat

Antigen fractions from adult S. mansoni, obtained from infected mice, were isolated by a variety of methods. A readily soluble fraction was obtained in good yield by freezing and thawing the schistosomes, while the less soluble residue was fractionated by the use of a number of the methods currently used for the extraction of tissue and cell surface antigens. The dialyzed, centrifuged products were characterized by acrylamide gel disc electrophoresis methods, agar gel precipitin reactions with antisera from rabbits immunized with whole schistosome homogenate, and by Prausnitz-Kustner (P-K) assay with sera from schistosome infected rats. The pattern of P-K reactivity suggested that there were a number of different antigen specificities involved in the reaginic antibody response to schistosome infection in rats. With repeated infection and increased duration of infection, more different antigens seemed to be involved in the reagin response. The schistosome antigen fraction obtained by freezing and thawing was especially reactive with both early infection rat sera and sera from multiply infected rats. Both the soluble fraction isolated by freezing and thawing and residue solubilized materials were found to be able to induce the formation of reagin antibodies on immunization with alum and B. pertussis vaccine.     

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.     

Anatomical localization of glucose uptake by Schistosoma mansoni adults

An apparatus was used to determine whether glucose enters adult Schistosoma mansoni through the oral sucker or the integumental surfaces. The results confirm the view that the integument is the primary site of glucose absorption in S. mansoni. Incubation of the parasites in 5-hydroxytryptamine (prior to insertion into the apparatus) depleted glycogen stores and increased glucose uptake. The demonstration of integumental uptake of glucose by female worms is consistent with the hypothesis that in copula the male schistosome provides glucose to the female.     

Is There a Sphingomyelin-Based Hydrogen Bond Barrier at the Mammalian Host–Schistosome Parasite Interface?

Schistosomes develop, mature, copulate, lay eggs, and live for years in the mammalian host bloodstream, importing nutrients across the tegument, but entirely impervious to the surrounding elements of the immune system. We have hypothesized that sphingomyelin (SM) in the parasite apical lipid bilayer is responsible for these sieving properties via formation of a tight hydrogen bond network with the surrounding water. Here we have used quasi-elastic neutron scattering for characterizing the diffusion of larval and adult Schistosoma mansoni and adult Schistosoma haematobium in the surrounding medium, under various environmental conditions. The results documented the presence of a hydrogen bond barrier around larvae and adult schistosomes. The hydrogen bond network readily collapses if worms are subjected to hypoxic conditions, likely via activation of the parasite tegument-associated neutral sphingomyelinase, and consequent excessive SM hydrolysis. The slower dynamics of lung-stage larvae as compared to adult worms has been related to the existence of hydrogen-bonded networks of different strength and then to their differential resistance to immune attacks.     

Functional Mapping of Protein Kinase A Reveals Its Importance in Adult Schistosoma mansoni Motor Activity

Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and ‘smart’ anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology.     

In vitro selection of drug resistant Schistosoma mansoni

Schistosomules of Schistosoma mansoni were cultured for 3 days in the presence of schistosomicides and then inoculated intraperitoneally into mice. Drug concentrations killing greater than 99.8% of schistosomules were amoscanate 0.1 p.p.m., oltipraz, 0.5 p.p.m., oxamniquine 240 p.p.m., praziquantel 8 p.p.m. Comparison of drug response of the unselected and selected strains as adult worms in mice showed an increase in tolerance to amoscanate, oltipraz and oxamniquine, but not praziquantel. The oxamniquine tolerant strain did not respond to oxamniquine at 500 mg kg⁻¹. The unselected strain increased in tolerance to three drugs during routine passage in the laboratory. Greater numbers of schistosomules derived from snails exposed to ethyl methane sulfonate appeared to survive culture in metrifonate, suggesting that it may be possible to produce drug resistant schistosomes by mutation and selection.