Ultrastructure, carbohydrate, and amino acid analysis of two preparations of the cercarial glycocalyx of Schistosoma mansoni.

This study determined the amino acid and carbohydrate composition of 2 cercarial glycocalyx preparations obtained after phenol-water extraction and subsequent gel chromatography. Labeled cercariae were subjected to 85% phenol, thereby dissociating the glycocalyx into the aqueous phase, which was dialyzed and chromatographed on Sepharose CL-2B or -4B in either 4 M guanidine hydrochloride (GuHCl) or 0.1% sodium dodecyl sulfate (SDS). The label eluted primarily in the void volume and was antigenic as tested with rabbit polyclonal antibodies by immunoblotting. Approximately 6 micrograms of protein and 28 micrograms of carbohydrate were obtained from 10(5) cercariae in the antigenic void volume fraction after SDS chromatography. Threonine, serine, and glutamic acid comprised 44% of the amino acid residues of the protein. The predominant sugar was fucose. Galactosamine, glucosamine, galactose, and mannose were also detected. After GuHCl chromatography, free amino acids, predominantly glycine and serine, comprised 17% of the total protein. The carbohydrate composition was similar to that of SDS-chromatographed extracts. Phenol-water extracts eluting in the void volume of Sepharose CL-2B were compared by negative staining and scanning electron microscopy with material obtained from medium in which cercariae shed glycocalyx during transformation to schistosomula. Both the isolated material and the transformation medium contained particles 20-50 nm in diameter, with subunits of 15-20 nm. These studies show that the cercarial glycocalyx is particulate, contains mainly carbohydrate and some protein, and is solubilized by phenol-water extraction.

Differential adhesive properties of the surface of human blood flukes Schistosoma mansoni. Sexual, regional, and developmental differences in interfacial free energies and structure of glycocalyx.

While infecting a vertebrate host, blood flukes (Schistosoma mansoni) must continually resist adhesions by immune effector cells. However, the male and female schistosomes must adhere to one another in order to establish and maintain the sexual pairing process after 4 wk postinfection. Using a contact angle method, the relative adhesiveness of male and female parasites were determined. Results indicate that schistosomes restrict effector cell adhesion through developmental, sexual, and regional differences in adhesive properties.

Identification of a sialic acid containing glycocalyx on the surface of Schistosoma mansoni.

Ruthenium red fixation of adult Schistosoma mansoni revealed the existence of a negatively charged layer external to the outer bilayer, which was morphologically similar to the glycocalyx of other cell types. Regional and sexual differences were found in the extent and organisation of the surface coat, which can be correlated with interfacial free energy, adhesiveness and protection from immune effectors. Neuraminidase treatment confirmed the presence of surface sialic acid. Mechanical or skin penetrated schistosomula, maintained in vitro for 24 h were found not to have a glycocalyx and this may relate to their increased susceptibility to immune killing. Lung stage schistosomula however, did bind ruthenium red to their surface.

Sequential removal of outer bilayer and apical plasma membrane from the surface epithelial syncytium of Schistosoma mansoni.

The outer and inner bilayers of the apical membrane complex of Schistosoma mansoni were sequentially stripped from adult worms by two incubations in 0.1% digitonin solutions. Membrane removal was evaluated by electron microscopy of worms and bilayer material, using Con A-ferritin as a marker for the outer bilayer. Amounts of Con A removed by the digests were measured with a tritiated Con A marker. To measure the purity of the fractions membrane markers were characterised and quantitated for both bilayers. In the absence of the usual enzymatic markers for plasma membrane diazotised [125I]-iodosulfanilic acid was used as a marker for the outer bilayer. Alkaline phosphatase and a Na+, Mg2+-ATPase were localised to the inner bilayer. From these results we can deduce that the inner bilayer is analogous to the typical, apical plasma membrane of other animal epithelia. The outer bilayer does not share these enzymatic similarities. The integrity of the syncytium after removal of the outer bilayer and the increased levels of lactate dehydrogenase in the supernatant after removal of the inner bilayer suggests that the outer bilayer is secondary in maintaining the permeability barrier of the apical membrane complex, with respect to soluble proteins. The possible significance of these results in terms of the destructive action of complement on the parasite are discussed.

Enrichment and partial enzyme characterization of ATPase activity associated with the outward-facing membrane complex and inward-facing membrane of the surface epithelial syncytium of Schistosoma mansoni.

The outward-facing (OFM) and inward-facing (IFM) membranes of the surface epithelial syncytium of Schistosoma mansoni were separated by sequential exposure to saponin solutions. The OFM, containing both inner and outer bilayers, contained ATPase activity that was stimulated by Mg2+ and Ma+, but not K+ or HCO-3, and was inhibited by Ca2+ and ethacrynic acid. The OFM enzyme was unaffected by ouabain, oligomycin, SCN- and azide and had a pH optimum of 7.5. The OFM ATPase therefore has properties similar to ATPases characterized from the apical membrane of a variety of epithelial cells where it is thought to augment the regulatory cell volume decreasing function of (Na++K+)Mg2+- ATPase. The IFM contained ATPase activity that was stimulated by Mg2+, Na+ and K+, and was inhibited by ouabain indicating the IFM enzyme was the Na+-pump ATPase. The results are discussed in terms of the transepithelial transport function of the surface epithelial syncytium and a Ca2+-ATPase reported previously from the OFM of S. mansoni.

Survival and behavior of Onchocerca gutturosa microfilariae in vitro.

Microfilariae of Onchocerca gutturosa were extracted from the skin of the bovine naval by teasing in saline and then suspending the skin in a gauze bag overnight in Tyrode's solution with 20% human serum. They survived for ten days in this medium, with antibiotics, in plastic microtitration plates at 11 C. After dispersal in flat containers, microfilariae slowly assumed clumped dispersion patterns. They also accumulated in glass wool, suggesting that aggregation was a tactile phenomenon. Analysis of videotapes of microfilarial movement indicated that activity, expressed as the number of undulations present on the body and the rate of knot formation (where the microfilaria held itself tightly coiled), increased up to 40 C and thereafter declined. The rate of headwave formation, however, appeared constant up to 40 C after which it declined. Where the greatest number of undulations were found on the body the wave amplitude was minimal. Because greater apparent activity was not correlated with increased forward progression, microfilariae at lower temperatures may travel farther than those at 40 C. This may relate to site preference in the host and to uptake by the vector Simulium ornatum.

Preparation and partial characterisation of a multilamellar body fraction from Schistosoma mansoni.

Multilaminate vesicles were purified, from homogenised whole Schistosoma mansoni adults, by differential density centrifugation on sucrose gradients, following methods previously employed for isolating multilamellar bodies (MLB) from mammalian lung. Morphometric analysis, on electron micrographs, of the pelleted fraction revealed that the pellet contained at least 56% MLB (by volume of solid material). An apparent projection core was described in both fixed MLB from our fraction and in unfixed MLB from a freeze thaw preparation of whole worms. The phospholipid-protein ratio of the MLB fraction was 1.6:1. The major phospholipid classes were separated and identified by quantitative, two dimensional, thin layer chromatography on silica gel. Phosphatidylcholine was the predominant phospholipid in the MLB fraction, comprising 57% of the total phospholipids. Phosphatidic acid phosphatase, previously reported from lung MLB but not schistosomes, was detected in the fraction. The high activity of this enzyme suggests a more active role for schistosome MLB than that of a mere reservoir of preformed membrane precursors.