The cercarial glycocalyx of Schistosoma mansoni

Cercariae, the freshwater stage of Schistosoma mansoni infectious to man, are covered by a single unit membrane and an immunogenic glycocalyx. When cercariae penetrate the host skin, they transform to schistosomula by shedding tails, secreting mucous and enzymes, and forming microvilli over their surface. Here the loss of the glycocalyx from cercariae transforming in vitro was studied morphologically and biochemically. By scanning electron microscopy, the glycocalyx was a dense mesh composed of 15-30 nm fibrils that obscured spines on the cercarial surface. The glycocalyx was absent on organisms fixed without osmium and was partially lost when parasites aggregated in their own secretions before fixation. By transmission electron microscopy, a 1-2 microns thick mesh of 8-15-nm fibrils was seen on parasites incubated with anti-schistosomal antibodies or fixed in aldehydes containing tannic acid or ruthenium red. Cercariae transformed to schistosomula when tails were removed mechanically and parasites were incubated in saline. Within 5 min of transformation, organisms synchronously formed microvilli which elongated to 3-5 microns by 20 min and then were shed. However, considerable fibrillar material remained adherent to the double unit membrane surface of schistosomula. For biochemical labeling, parasites were treated with eserine sulfate, which blocked cercarial swimming, secretion, infectivity, and transformation to schistosomula. Material labeled by periodate oxidation and NaB3H4 was on the surface as shown by autoradiography and had an apparent molecular weight of greater than 10(6) by chromatography. Periodate-NaB3H4 glycocalyx had an isoelectric point of 5.0 +/- 0.4 and was precipitable with anti-schistosomal antibodies. More than 60% of the radiolabeled glycocalyx was released into the medium by transforming parasites in 3 h and was recovered as high molecular weight material. Parasites labeled with periodate and fluorescein-thiosemicarbazide and then transformed had a corona of fluorescence containing microvilli, much of which was shed onto the slide. Material on cercariae labeled by lodogen-catalyzed iodination was also of high molecular weight and was antigenic. In conclusion, the cercarial glycocalyx appears to be composed of acidic high molecular weight fibrils which are antigenic and incompletely cleared during transformation.     

Proteomic analysis of Schistosoma mansoni cercarial secretions

Schistosomiasis is a global health problem caused by several species of schistosome blood flukes. The initial stage of infection is invasion of human skin by a multicellular larva, the cercaria. We identified proteins released by cercariae when they are experimentally induced to exhibit invasive behavior. Comparison of the proteome obtained from skin lipid-induced cercariae (the natural activator), a cleaner mechanical induction procedure, and an uninduced proteomic control allowed identification of protein groups contained in cercarial acetabular gland secretion versus other sources. These included a group of proteins involved in calcium binding, calcium regulation, and calcium-activated functions; two proteins (paramyosin and SPO-1) implicated in immune evasion; and protease isoforms implicated in degradation of host skin barriers. Several other protein families, traditionally found as cytosolic proteins, appeared concentrated in secretory cells. These included proteins with chaperone activity such as HSP70, -86, and -60. Comparison of the three experimental proteomes also allowed identification of protein contaminants from the environment that were identified because of the high sensitivity of the MS/MS system used. These included proteins from the intermediate host snail in which cercariae develop, the investigator, and the laboratory environment. Identification of proteins secreted by invasive larvae provides important new information for validation of models of skin invasion and immune evasion and aids in rational development of an anti-schistosome vaccine.     

Glycomics analysis of Schistosoma mansoni egg and cercarial secretions

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.     

Schistosoma mansoni: ultrastructure of cercarial escape glands

While in the sporocyst, the cercaria of Schistosoma mansoni has a pair of unicellular escape glands whose funduses are located together on one side of the body. The funduses taper into microtubule lined ducts that open on the anterior surface of the oral sucker through desmosome supported pores in the immediate vicinity of the ducts of the acetabular glands. The glands contain membrane-bound secretory granules which have a fine medium-dense, homogeneously granular material in their matrices. A large membrane-bound reservoir of granular material, whose texture is similar to that of the secretory granules, is often seen in the cytoplasm of the funduses. In free-swimming cercariae, the ducts in the oral sucker are the only obvious remains of the escape gland.     

Schistosoma mansoni: development of the cercarial glycocalyx

The development of the cercarial glycocalyx of Schistosoma mansoni was studied by transmission electron microscopy and immunofluorescence light microscopy employing antibodies raised against extracted and chromatographed glycocalyx. By electron microscopy, cercariae present in the brood chamber of daughter sporocysts were surrounded by an electron-dense granular and fibrillar matrix. This material appeared structurally distinct from the glycocalyx which was coarsely fibrillar and located only on the surface of organisms that had developed a final tegument. The thickness of the glycocalyx apparently increased with the maturation of the tegument, since teguments that had many spines also had the thickest glycocalyx. Immunofluorescent staining of frozen sections of infected snail hepatopancreas showed that glycocalyx antigens were present on the surface of the cercariae and not in the matrix within the brood chamber or in snail tissues. Immunofluorescent staining of isolated larval cercariae showed staining of some but not all parasites with partially elongated tails. These studies suggest that the glycocalyx develops late in cercarial development (late in Stage 6 or in Stage 7 of Cheng and Bier), is made by the cercariae themselves, and is not a product of either the sporocyst wall cells or snail hepatopancreas.     

Schistosoma mansoni: analysis of cercarial transformation methods

Four methods of transforming cercariae to schistosomulae in vitro in ELAC buffer (pH 7.2, 37 C, 0-6 hr incubation) were compared in relation to biochemical and ultrastructural characteristics. The transformation methods used were chemical (3 mM linoleate), mechanical (centrifuge/vortex), mechanical/chemical, and heat (incubation at 37 C). Ultrastructural characteristics examined were based on the presence or absence of glycocalyx, heptalaminate membrane, cyton granules, and nuclear condition. Two EM fixation methods were used. Biochemical parameters assayed were loss of water tolerance (uptake of trypan blue dye), eicosanoid biosynthesis (PGE, LTB4, and 5-HETE), protein synthesis (leucine uptake), RNA synthesis (uracil and orotic acid uptake), and DNA synthesis (thymidine uptake). EM characteristics were remarkably similar for all transformation methods except heat incubation, with transformed cercariae evidencing the characteristics of schistosomulae (cyton granule migration, absence of glycocalyx and heptalaminate membrane); however, euchromatic nuclei could not be demonstrated using in vivo or in vitro transformation methods. Despite the ultrastructural similarities between transformation methods, biochemical data demonstrated that the resultant organisms were quite different. The chemical transformation method gave the highest rate of loss of water tolerance and eicosanoid production. RNA and protein synthesis were not correlated to ultrastructural changes and were highest in those organisms undergoing mechanical transformation methods, significantly higher than in those cercariae transformed by the chemical method. DNA synthesis was not demonstrated using any transformation method, although thymidine uptake did occur. Our data indicate substantial biochemical differences exist between morphologically similar organisms. Thus, experiments using any type of artificially transformed schistosomule must be interpreted with caution until additional biochemical and physiological studies on cercarial transformation are undertaken.     

Schistosoma mansoni: immunogenic glycoproteins of the cercarial glycocalyx

Immunochemical studies at the level of the light and electron microscope showed that a monoclonal antibody, 128C3/3, was directed to an epitope in the glycocalyx of Schistosoma mansoni cercariae. Immunoprecipitation of surface labeled cercarial extracts with this monoclonal antibody demonstrated that the glycocalyx is composed of at least five components, including a very large molecular size polypeptide and polypeptides of 220, 180, 170, and 15 kDa. After transformation of cercariae to schistosomula, these polypeptides were shed from the surface and were therefore no longer accessible to surface labeling. Monoclonal antibody 128C3/3 was also reactive with a 38 kDa polypeptide from schistosomula; this polypeptide was weakly expressed on the surface of cercariae. Analysis of immunoprecipitates of radioiodinated protein extracts of cercariae, newly transformed schistosomula, and 36 hr in vitro cultured schistosomula showed that the 180 and 170 kDa polypeptides continued to be expressed within the organism following transformation, but were not accessible to surface labeling. Lectin binding studies revealed differences in the oligosaccharide composition of the six polypeptides. With the exception of the 15 kDa antigen, all the polypeptides reactive with 128C3/3 were highly immunogenic in infected mice and humans.     

Schistosoma mansoni: fine structure of cercarial acetabular glands

Emerged cercariae of Schistosoma mansoni were studied with the electron microscope for the purpose of describing the acetabular (penetration) glands and their cellular investment. The two pre- and three postacetabular unicellular glands consist of enlarged aboral areas (funduses) and their oral extensions as ducts. The glands were morphologically similar except for their shape and secretory globules. In the funduses of the postacetabular glands the globules were of a single type, spheroidal to irregular in shape, with numerous electron-dense areas. Preacetabular secretory globules appeared to be of several types, varying in size, shape, and homogeneity. Some were of uniform density; others showed electron-lucid areas. The fine structure of both pre- and postacetabular globules changed as they were compared in the funduses, in progressively oral areas of the ducts and after extrusion. These changes were thought to be transitional. Microtubules and close cellular investiture of the glands by muscle, nerve, and unclassified cells with extended interwoven processes appeared to provide structural support.     

Schistosoma mansoni: cercarial eicosanoid production and penetration response inhibited by esculetin and ibuprofen

Cercariae of Schistosoma mansoni produce a wide variety of eicosanoids when stimulated by 3.3 mM linoleate. High-performance liquid chromatography indicated that 10(-5) M esculetin dramatically decreased eicosanoid production by cercariae. Ibuprofen (10(-4) M) also decreased eicosanoid production, but to a lesser extent. These results were confirmed by radioimmunoassay using time-dose curves for esculetin and time curves for ibuprofen. The results reported here indicated that, for cercariae, ibuprofen was neither a specific cyclo-oxygenase inhibitor, as has been reported for platelet and endothelial cells, nor was esculetin a specific inhibitor of lipoxygenase, as has been reported for platelets and mastocytoma cells. Rather, both drugs inhibited cyclo-oxygenase and lipoxygenase enzyme systems. Further, the data indicated that two forms of cyclo-oxygenase exist in cercariae (isozymes?), one initiating the conversion of gamma-dihomolinolenate into the 1-series prostaglandins and another acting on arachidonate forming the 2-series prostaglandins. The cyclo-oxygenase acting on arachidonate has a greater sensitivity to both ibuprofen and esculetin than the enzyme acting on gamma-dihomolinolenate. Cercarial lipoxygenases also varied greatly in their sensitivity to ibuprofen.     

The human blood fluke Schistosoma mansoni synthesizes glycoproteins containing the Lewis X antigen.

Infection of vertebrates with the parasitic blood fluke Schistosoma mansoni induces a variety of host immune responses, which are directed against both protein and carbohydrate antigens. In this report, we describe our studies on the structures of antigenic oligosaccharides derived from glycoproteins synthesized by S. mansoni. Immobilized antibodies derived from the sera of infected hamsters and mice bind to a family of high molecular weight Asn-linked oligosaccharides in glycoproteins from the adult parasite. Structural analysis of the major antigenic oligosaccharides revealed that they have high amounts of fucose-linked alpha 1,3 to N-acetylglucosamine residues within the linear repeating disaccharide (3Gal beta 1-4GlcNAc beta 1)n, a poly-N-acetyllactosamine sequence containing the Lewis X antigenic blood group. The remarkable ability of S. mansoni to synthesize these vertebrate-type oligosaccharides may have implications in both the mechanisms of host-parasite interactions and on the development of vaccines to prevent this disease in humans.     

Selective expression of different fucosylated epitopes on two distinct sets of Schistosoma mansoni cercarial O-glycans: identification of a novel core type and Lewis X structure.

The glycobiology of Schistosoma mansoni is dominated by developmentally regulated expression of various fucosylated structures, most notably the Lewis X epitope and a multifucosylated sequence, Fuc alpha1-->2Fuc alpha1-->, in its various forms. For the infective cercarial stage, Lewis X has been structurally identified on glycosphingolipids and N-glycans of total glycoprotein extracts, and a population of multifucosylated glycoproteins were found to carry a unique terminal sequence, +/-Fuc alpha1-->2Fuc alpha1-->[3GalNAc beta1-->4(Fuc alpha1-->2Fuc alpha1--> 2Fuc alpha1-->3) GlcNAc beta1-->3Gal alpha1-->](n), on their O-glycans. Using a mass spectrometry approach coupled with chromatographic separation, sequential exoglycosidase digestion, periodate oxidation, and other chemical derivatization, we demonstrate that Lewis X could also be carried on the cercarial O-glycans, but the two distinctive sets of fucosylated epitopes were conjugated to two different core structures. Lewis X, lacNAc, or single GlcNAc was found to attach directly to the -->3Gal beta1-->3GalNAc core and indirectly via another beta-Gal residue branching off from C6 of the reducing end GalNAc to give a biantennary-like structure. The -->3(+/-Gal beta1-->6)Gal beta1-->3(-->3Gal beta1-->6)GalNAc core thus characterized represents a novel core type for O-glycans. In contrast, the previously characterized multifucosylated terminal sequences were carried on conventional type 1 and 2 cores. The smallest structures of the reductively released O-glycans were defined as GalNAc beta1-->4GlcNAc beta1-->3Gal beta1-->3GalNAcitol with a total of two to four fucoses attached to the terminal lacdiNAc. alpha-Galactosylation of the nonreducing terminal beta-GalNAc instead of fucose capping leads to further elongation with another lacdiNAc unit that could also extend directly from C6 of the reducing end GalNAc and similarly elongated or terminated.     

Schistosoma mansoni: relationship between cercarial production levels and snail host susceptibility

Two populations of Biomphalaria glabrata snails differing slightly in their susceptibility to Schistosoma mansoni infection showed dramatic differences in cercarial output per snail. Exposed to five or more miracidia, snails from a group with a 90-100% susceptibility rate (Group A) produced nearly twice the number of cercariae as those from a group with a 70-80% susceptibility rate (Group B). Exposure of individual snails to known numbers of miracidia resulted in higher numbers of primary (mother) sporocysts in Group A snails than in Group B snails. However, monomiracidial exposure of snails from both groups resulted in equivalent numbers of cercariae produced per positive snail, indicating that, once established, all primary sporocysts possess a similar reproductive potential. Morphometric analysis of serially sectioned 9-day-old primary sporocysts supported this conclusion; the size of the primary sporocysts and the size and numbers of secondary (daughter) sporocysts within each primary sporocyst were comparable in snails from both groups. The data indicate cercarial production in this system is regulated prior to, and/or during, early development of the primary sporocyst.     

Comparative analysis of the Schistosoma mansoni and Schistosoma bovis cercarial production under the influence of praziquantel

Snails infected with Schistosoma mansoni and S. bovis and fed with a food-Praziquantel mixture stop shedding cercariae for several days. The cercarial production restarts at different levels after stopping treatment: for some Biomphalaria glabrata, the restarting phase of production reaches a high level whereas for other B. glabrata and all of the P. metidjensis, the level of production remains low. Histological studies revealed that at the exact moment of this treatment, there is a total destruction of many nearly mature cercariae whereas the young cercarial embryos and the tegument of the sporocyst remain unharmed. When treatment stops, there is a pronounced proliferation of third-generation sporocysts (Sp III) which invade the available space of the snails' digestive gland.     

Schistosoma mansoni: salicylanilides as topical prophylactic against cercarial penetration of mice

Salicylanilide and 37 of its analogs were applied topically to mice as candidate chemoprophylactic agents against Schistosoma mansoni cercarial penetration. The compounds were solubilized in absolute methanol, dimethyl sulfoxide, or isopropanol at concentrations not exceeding 1.25% W/V. The tails of the mice of each experimental group were treated by immersion for 5 min in the test compound solution or in the vehicle. The treated tails of 5 mice from each group were washed for 30 min in flowing tap water 3-4 hr after compound application. Tails of all mice were then exposed to approximately 100 cercariae by tail immersion for 1 hr, 24 hr after treatment. The portal veins were perfused 49 days after exposure and worm burdens were determined. The protective capacity of each compound was calculated by comparing the reduction of the mean worm burdens of the compound treated mice to the worm burdens of those treated with only the vehicle and expressing the resulting value as percentage protection. Of the 38 compounds tested, 20 provided 98% or better protection if the treated tails were not washed before exposure to cercariae. Of these 20 active compounds, 16 provided 98% or better protection from infection by S. mansoni cercariae even after the mouse tails were subjected to the 30 min wash.     

Schistosoma mansoni: complement and cercarial tail loss during in vitro transformation.

When Schistosoma mansoni cercariae are incubated at 37 C in media containing serum, the organisms lose their tails and change into viable, infective schistosomula. Tail loss does not occur in the absence of serum, or when the serum is heat inactivated. In the present studies, tail loss during in vitro conversion was shown to be complement dependent. The capacity of fresh serum to promote tail loss was markedly suppressed or abolished by cobra venom factor, zymosan, Sepharose CL-4B AND anti-C3 antibody. The alternative rather than the classic complement pathway appeared to be responsible since (1) binding of anti-C3 to cercariae required magnesium, but not calcium; (2) both C4-deficient serum and C2-deficient serum supported tail loss; but (3) human serum heated to 50 C for 20 min to inactivate Factor B did not support tail loss. Cercarial tail loss also required the terminal complement components C5 through C8. The extent and rate of tail loss was normal in agammaglobulinemic sera indicating that the complement effect was not antibody dependent.