Eugene G. Hayunga

Surface antigens of Schistosoma mansoni

Abstract The outer tegument membrane of 18 h artificially prepared schistosomula of Schistosoma mansoni was labelled using the non-permeant diazonium salt of [ 125 I]iodosulphanilic acid. Eight iodinated surface proteins were identified by SDS-polyacrylamide gel electrophoresis. Three of these proteins, one of which is glycosylated, can be precipitated by immune serum.

ISOLATION AND CHARACTERIZATION OF SURFACE ANTIGENS FROM SCHISTOSOMA MANSONI. I. EVALUATION OF TECHNIQUES FOR RADIOISOTOPE LABELING OF SURFACE PROTEINS FROM ADULT WORMS

Radiolabeled surface proteins of adult Schistosoma mansoni were prepared by in vitro labeling of whole worms, and by labeling freeze-thaw surface membrane extracts. Incorporation of 125I into surface proteins was attempted using the lactoperoxidase, chloramine-T, iodosulfanilic acid, and Bolton-Hunter methods. Radiolabeling of whole worms with lactoperoxidase, chloramine-T and iodosulfanilic acid yielded a single protein peak (mol wt greater than 100,000) on SDS-PAGE, and showed considerable incorporation of label in the lipid fraction. Bolton-Hunter labeling of whole worms yielded four major peaks with molecular weights of 100,000, 60,000, 30,000 and 21,000, and minor peaks with molecular weights of 26,000, 36,000, 43,000, 68,000 and 78,000; three of the four major peaks corresponded to prominent bands in Coomassie blue-stained gels. Although carbohydrate-labeling techniques were not successful, a single carbohydrate band, molecular weight greater than 100,000, was detected was PAS staining. Radiolabeling of freeze-thaw extracts yielded results similar to those obtained with whole worms. Electron microscopy revealed the tegument to be left intact and undamaged after labeling with the Bolton-Hunter reagent.

CHARACTERIZATION OF SURFACE GLYCOPROTEINS ON SCHISTOSOMA MANSONI ADULT WORMS BY LECTIN AFFINITY CHROMATOGRAPHY

Adult Schistosoma mansoni were radiolabeled by direct radioiodination using the Bolton-Hunter reagent or by metabolic labeling using radioactive hexose precursors. Tegumental material was extracted by freeze-thaw or by incubation in the non-ionic detergent Nonidet P-40, then applied to chromatography columns containing the following immobilized lectins: Con A, lentil lectin, wheat germ agglutinin, soybean agglutinin and the agglutinins from Ricinus communis and Helix pomatia. SDS-PAGE analysis of the sugar eluates from these columns revealed the presence of 15 glycoproteins with apparent molecular weights greater than or equal to 300,000, 215,000, 168,000, 152,000, 134,000, 122,000, 108,000, 83,000, 58,000, 53,000, 46,000, 41,000, 34,000, 30,000 and 23,500. Many of the glycoproteins reacted with more than one lectin. Information about carbohydrate content and lectin binding provides a preliminary characterization of the tegumental glycoprotein antigens of adult worms.

Morphological adaptations of intestinal helminths

Nematodes, trematodes, cestodes, and acanthocephalans each have become adapted in different ways to the microenvironment of the vertebrate intestine. Life in this specialized habitat affords parasites a reliable source of nutrients, a relatively homeostatic environment, and protection from predators but, in exchange for these advantages, presents the special challenges of exposure to digestive enzymes, normal peristalsis, and host immune response to infection. Logically, the surface of the parasite should be the first part of the organism to encounter such challenges, and, for this reason, any response or reaction by the parasite is expected to be manifested at the parasite-host interface. Morphological adaptations of intestinal helminths to their microenvironment include modification of the tegumental surface that affords protection and increases absorptive surface area, development of specialized attachment organs, and, in some cases, complete loss of their own internal digestive system. Representative examples of such adaptations by helminths are described and discussed in terms of the parasites nutritional requirements, site selection, and host specificity, and the possibility is suggested that some helminths may have adapted in ways that exploit host defensive mechanisms for their own benefit.

Isolation and characterization of surface antigens from Schistosoma mansoni. II. Antigenicity of radiolabeled proteins from adult worms.

Adult Schistosoma mansoni were radiolabeled in vitro with 125I Bolton-Hunter reagent. Surface membrane antigens were solubilized with non-ionic detergent, then reacted with infection or normal serum. The antigen-antibody complexes were then precipitated with staphylococcal protein A immunoadsorbent, eluted with urea and SDS, and fractionated by SDS-PAGE. The results indicated the presence of 6 to 8 tegument antigens, depending on the type of antisera used. Human antisera to S. japonicum and S. haematobium reacted with some but not all of the antigens identified with human S. mansoni infection serum; this implies the presence of species-specific tegument antigens. The molecular weights of the radiolabeled antigens ranged from 10,000 to 100,000. A large (greater than 100,000) molecular weight glycoprotein and an uncharacterized lipid fraction appeared to be precipitated nonspecifically. Immunoprecipitation methods with anti-mouse IgG and anti-mouse whole serum failed to detect the presence of hostlike antigens in the labeled extracts. Several of the labeled proteins from S. mansoni were found to react with serum from patients infected with either S. haematobium or with S. japonicum.

Some problems associated with radiolabeling surface antigens of helminth parasites: A brief review

Recent developments in technology have facilitated substantial advances in the characterization of surface antigens from a wide variety of both normal and neoplastic cells. However, the immunochemistry of parasites has lagged behind. Efforts to apply conventional radiolabeling methods to helminths have not always been successful. Experimental work with Schistosoma mansoni is reviewed to illustrate common problems encountered in surface labeling studies. These findings should provide insight for the future investigation of other helminth species.

Evidence for selective incorporation of host immunoglobulin by strobilocerci of Taenia taeniaeformis.

Strobilocerci of Taenia taeniaeformis were obtained from laboratory rats 90 days after experimental infection. Cyst fluid, whole parasite homogenate, and rat serum each were fractionated by SDS-PAGE, immobilized on nitrocellulose by western blot, and probed with conjugated goat anti-rat IgG. Reactive bands with relative mobilities corresponding to rat IgG were found in all 3 samples. Additional bands in cyst fluid and parasite homogenate may represent enzymatic degradation of IgG. The pattern of reactive bands in the homogenate discounts the nonspecific adsorption of host molecules onto the tegument and suggests selective incorporation of serum proteins. The presence of an IgG-like molecule of atypical molecular weight is consistent with either molecular mimicry or enzymatic cleavage of IgG bound to the tegument. The relevance of serum protein utilization by the parasite to evasion of the host immune response is discussed.

Expression of lectin-binding surface glycoproteins during the development of Schistosoma mansoni schistosomula.

Tegumental glycoproteins of Schistosoma mansoni cercariae, mechanically produced 24-hr and 48-hr schistosomula, and adult worms were radioiodinated with the Bolton-Hunter reagent, then isolated by lectin affinity chromatography. SDS-PAGE revealed Con A binding glycoproteins with apparent molecular weights of 180,000, 150,000, 43,000, and 30,000 in detergent extracts of the tegument of cercariae. These glycoproteins are retained by 24-hr mechanically produced, cultured schistosomula and are accompanied by the appearance of 2 additional labeled glycoproteins, mol. wt. 66,000 and 57,000. In 48-hr schistosomula, there is a marked increase in the relative size of the 66,000 mol. wt. peak. In contrast, the 57,000 mol. wt. glycoprotein is the major radiolabeled Con A binding component of the adult tegument; the other peaks are either reduced or absent in adults. Similar findings were obtained following affinity chromatography using immobilized Lens culinaris lectin or Ricinus communis agglutinin, and following metabolic labeling of glycoproteins with tritiated galactose.

Early diagnosis of Schistosoma mansoni in mice using assays directed against cercarial antigens isolated by hydrophobic chromatography.

Two diagnostic assays are described for the early diagnosis of acute schistosomiasis, using a defined cercarial antigen preparation obtained by hydrophobic chromatography. Circulating IgM antibodies against this antigen fraction could be detected by ELISA as early as 1 wk after exposure in experimentally infected mice; IgM levels against other antigens and IgG levels against all the preparations examined were not significantly elevated until approximately 4-5 wk postinfection. Circulating antigen was detected as early as 3 days after exposure by a competitive inhibition ELISA using rabbit serum prepared against the cercarial antigen; antigen levels in the serum of mice with a 100-worm infection were found to exceed 100 ng/ml. Studies using sera from infected humans indicate that the assay can also recognize chronic S. mansoni, S. haematobium or S. japonicum infections. In a very limited field study, the specificity of the circulating antigen assay with regard to other helminthic infections was found to be 85%; sensitivity 100%. Preliminary characterization of the relevant antigen indicates that it is a relatively hydrophobic polypeptide with a molecular weight of approximately 41,000 daltons. The implications of these findings with regard to the treatment of travelers or the conduct of seroepidemiological studies in endemic areas are discussed.

Partial characterization of radiolabelled antigens from adult Schistosoma haematobium.

Previously, Bolton-Hunter labeled adult S. mansoni proteins were fractionated by SDSPAGE to determine molecular weights and immunoprecipitated using human infection sera to detect cross-reactivity (Hayunga et al., 1979, J. Parasitol. 65: 488-496; 497-506). These observations are now extended to S. haematobium. In order to provide a direct comparison, the conditions of radiolabeling, detergent extraction and molecular weight determination were kept identical to those of the previous study. Cercariae from an Egyptian strain of S. haematobium in naturally infected Bulinus truncatus were used to infect golden hamsters in the laboratory. (We are grateful to Drs. P. LoVerde, K. Murrell, and V. Schinski for collecting infected snails in the field, and to Miss K. Groover, Mr. D. Olson, and K. Holman for maintaining the life cycle in the laboratory.) Approximately 20 to 30 adult worms, obtained by portal perfusion of 12-wk infected hamsters, were radiolabeled with 0.5 mCi 125I-Bolton-Hunter reagent as described previously (Hayunga et al., loc. cit.); the worms were motile and appeared undamaged after this procedure. Tegumental proteins were solubilized in 0.5% NP-40 for 15 min at 37 C, and the lysate run directly on SDS gels or immunoprecipitated as follows: Aliquots (approx. 100,000 c.p.m.) of labeled lysate were incubated overnight at 4 C with 25 ,ul serum, followed by incubation for 2 hr with 500 ,ul of a 10% w/v suspension of staphylococcal protein A (Pansorbin?, Calbiochem); after washing 3 times with BBS, samples were eluted with 4% SDS-6 M urea and fractionated by SDS-PAGE on 10% tube gels. Serum from patients infected by either S. haematobium or S. mansoni was provided by Dr. E. Higashi, NAMRU-3, Cairo; rabbit serum was prepared by subcutaneous inoculation of a freeze-thaw preparation of adult S. mansoni. Fractionation of radioiodinated S. haematobium proteins on SDS gels revealed heavily labeled peaks of >100,000, 60,000, 30,000, and 13,000 mol. wt., and minor peaks of 85,000, 43,000, 36,000, 33,000, 25,000, 23,000, and 18,000 mol. wt. (Fig. 1A). Label also occurred in two peaks, presumed to contain lipid material, that migrated with and slightly ahead of the dye front. When samples were electrophoresed under reducing conditions using dithiothreitol, the large (>100,000) molecular weight peak dissociated but no additional peaks were found (Fig. 1B). In order to detect cross-reactivity, the labeled lysate from