Flagellar membrane and paraxial rod proteins of Leishmania: characterization employing monoclonal antibodies.

Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family (Endotrypanum, trypanosoma, Leishmania) suggesting that these molecules may be evolutionarily conserved.

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.

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.

Evidence that a protective membrane epitope is involved in early but not late phase immunity in Schistosoma mansoni.

An anti-egg monoclonal antibody E.1, which is partially protective in passive transfer experiments, is shown in this study to recognize a membrane epitope on cercariae, schistosomula, and the ciliary plates of miracidia. E.1 did not bind to the surface membranes of lung or adult worms, or recognize secreted egg antigen in infected liver tissue. The E.1 epitope was present in the glycocalyx of cercariae, as well as on the syncytial membrane as determined by electron microscopy. Immunoprecipitation of iodinated surfaces of cercariae and schistosomula demonstrated E.1 binding to a high m.w. moiety in cercariae, which corresponds to the glycocalyx because it was not immunoprecipitated from schistosomula. In addition, a band at 38,000 daltons was immunoprecipitated from both cercariae and schistosomula. When compared with in vitro cultured parasites, schistosomula that were obtained from mice 1 to 24 hr after tail vein injection showed significant loss of E.1 binding. Consistent with the rapid loss of antigen in vivo, E.1 antibody was unable to passively transfer protection to naive mice when administered 5 days after cercarial challenge.

Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components.

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.

Human erythrocytes adhering to schistosomula of Schistosoma mansoni lyse and fail to transfer membrane components to the parasite.

We studied the adherence of human erythrocytes to larvae of the intravascular parasite Schistosoma mansoni by transmission microscopy, freeze fracture, and fluorescence techniques. In addition, we used the adherent cells to investigate the problem of host antigen acquisition. Schistosomula were cultured for from 24 to 48 h after transformation in order to clear the remnants of the cercarial glycocalyx. In some cases, the worms were preincubated with wheat germ agglutinin to promote adherence of the erythrocytes. The results were similar with and without the lectin except that more cells attached to the lectin-coated parasites. Erythrocytes adhered within a few hours and, unlike neutrophils, did not fuse with the parasite. A layer of 10-20-nm electron dense material separated the outer leaflets of the tegumental and plasma membranes. In addition, many deformed and lysed cells were seen on the parasite surface. The ability of the worm to acquire erythrocyte membrane constituents was tested with carbocyanine dyes, fluorescein covalently conjugated to glycophorin, monoclonal antibodies against B and H blood group glycolipids, and rabbit alpha-human erythrocyte IgG. In summary, glycophorin, erythrocyte proteins, and glycolipids were not transferred to the parasite membrane within 48 h. Carbocyanine dyes were rapidly transferred to the parasite with or without lectin preincubation. Thus, the dye in the worm membrane came from both adherent and nonadherent cells. These studies suggest that, in the absence of membrane fusion, the parasite may acquire some lipid molecules similar in structure to host membrane glycolipids by simple transfer through the medium but that B and H glycolipids and erythrocyte membrane proteins are not transferred from adhering cells to the worm.