Schistosoma mansoni: purification and characterization of the major acidic proteinase from adult worms.

We report purification of the major digestive proteinase from adult worms of Schistosoma mansoni. This enzyme is a thiol proteinase with a pH optimum of 5 and is activated by thiol reagents. It was purified 300-fold using a combination of gel chromatography and chromatofocusing. It readily hydrolyzed hemoglobin with an apparent Km of 0.29 microM and a specific activity of 27 micrograms degraded/min/mg enzyme at 37 C. Peptides with positively charged amino acids were preferentially cleaved. The enzyme degraded Boc-Arg-Arg-7-amino-4-methyl coumarin with a kcat/Km of 9083 M-1 sec-1. Lengthening the peptide chain to 3 amino acids or substituting glycine for the amino terminal arginine resulted in decreased activity. The enzyme was inhibited by chloromethylketone-derivatized peptides of similar sequence and by leupeptin. The purified proteinase exhibits microheterogeneity in different preparations with forms ranging in molecular weight from 30,000 to 35,000, and pI 5.7-6.0.

Enzymes of uridine 5'-monophosphate biosynthesis in Schistosoma mansoni.

In Schistosoma mansoni, the major product of in vitro orotate metabolism was orotidine 5'-monophosphate (OMP), whereas in mouse liver it was UMP. In contrast to mammalian cells, OMP appeared not to be 'channeled' from orotate phosphoribosyltransferase to OMP decarboxylase in S. mansoni, resulting in substantial degradation of OMP to orotidine. Significant differences were observed in the inhibitor specificity of phosphoribosyltransferase between S. mansoni and mouse liver, indicating that this enzyme may be a potential chemotherapeutic target in S. mansoni. Two distinct phosphoribosyltransferases were found in S. mansoni. One enzyme, having the higher molecular weight, utilized orotate, 5-fluorouracil and uracil as substrates, while the other only orotate. Both enzymes were inhibited by 5-azaorotic acid (oxonic acid) but only the 'orotate-specific' enzyme was inhibited by 4,6-dihydroxypyrimidine. OMP decarboxylase activity co-eluted with both phosphoribosyltransferases from Sephadex G-100 gel chromatography. We suggest that phosphoribosyltransferase in S. mansoni plays a role in both de novo UMP biosynthesis as well as in the salvage of uracil and uridine.

Host heme biosynthesis and degradation in schistosomiasis.

In mice infected with Schistosoma mansoni, hepatic heme metabolism is markedly altered. The production of the immediate precursor, delta-aminolevulinic acid, is diminished, while the activity of the catabolic enzyme, heme oxygenase, is greatly increased. These changes are accompanied by a reduction in the cellular content of hemoproteins in various organs. Specifically, cytochrome levels in myocardial mitochondria are reduced, and liver cytochromes P-450 and b5 are also diminished. As a consequence of the latter, the microsomal oxidative enzyme activities, which are mediated by P-450, such as ethylmorphine N-demethylase and aniline hydroxylase, are considerably impaired. Barbiturate-induced sleeping time in mice heavily infected with schistosomes was found to be significantly prolonged. A green discoloration of the liver and spleen seen in advanced murine schistosomiasis is not likely to be due to the production of abnormal pyrrolic pigments, since hemoglobin heme was found to be degraded via the usual catabolic pathways to physiological bile pigments. Total serum iron was found to be increased by 100% in schistosome-infected mice. Serum unsaturated iron binding capacity was, however, not increased significantly. Demonstration that the activities of enzymes of heme metabolism, which are known to be regulated by heme and metal ions, are altered in the host as a consequence of the parasitism suggests that these perturbations may be mediated by heme or its iron released by the digestion of erythrocytes by schistosomes.

Hemoglobinolytic activity of serum in mice infected with Schistosoma mansoni.

Sera from mice infected with Schistosoma mansoni were found to contain substantial amounts of an acid-active hemoglobinolytic enzyme. Recovery of this enzyme from aliquots of pooled 12-week infected serum, using a phenylalanine-agarose affinity column, showed that a portion of the enzyme binds tightly to the column at pH 4.0, and can be eluted with 0.01 M formic acid. Another larger portion of hemoglobin-digesting activity is not bound to the column and emerges with the buffer front. Sera from rats which were exposed to cercariae, but in whom worms were stunted and did not develop to maturity, were found not to contain hemoglobin-active protease. At the present time, the source of the enzyme has not been unequivocably proven. The enzyme found in the serum binds to and releases from the affinity column in the same manner as does hemoglobinase recovered from freeze-dried S. mansoni worms. Maximal activity of both enzymes against the substrate occurs at pH 4.5-5.0. Present evidence suggests that the protease present in the serum is of worm origin. It is postulated that this protein may be excreted by the parasite during the process of regurgitation of gut contents. Presence of worm enzyme circulating in the host plasma would correlate with the known sensitization of the host to schistosomal hemoglobin-digesting enzyme.

An analysis of allergy, immunoglobulin E, and diagnostic skin tests in schistosomiasis.

Factors governing the sensitization or desensitization of basophils and mast cells are discussed. Mathematical models are proposed which illustrate the effects of rising or falling specific or non-specific IgE titres on the tendency of these cells to degranulate. The models presented are consistent with the hypothesis that fine-tuning of the degranulatory event is achieved by one or more of the following mechanisms: alteration of the number of IgE receptors on the mast cell membrane; displacement of specific anti-schistosomal IgE by anti-other-IgE molecules; clipping or otherwise inactivating mast cell-fixed specific IgE receptor sites so as to render these incapable of binding antigen. Mechanisms proposed may explain how a mast cell population may evolve from highly sensitive to non-reactive allergic states during early and/or chronic periods of schistosomiasis, only to revert to highly sensitive states once again, after the disease has been overcome.

Structure of the schistosome eggshell: amino acid analysis and incorporation of labelled amino acids.

Preparations of eggshells of Schistosoma mansoni and S. japonicum were hydrolyzed and analyzed for amino acid composition. Both species showed great similarities in the proportions of each residue found. The predominant amino acid in shell hydrolysates was found to be glycine, which accounted for 37% of S. mansoni and 45% of S. japonicum amino acids. Four components (glycine, aspartic acid, lysine, and serine) totalled 68--75% of amino acids in the eggshells. Other individual amino acids were present in relatively small proportions ranging from 5.2--0.01%. Less than 1% of the amino acid residues were identified as tyrosine, and bityrosine was detected at a level not exceeding 1 in 1,600 residues. Carbohydrates were estimated to comprise 7.5--10% of the eggshell weight, based on hexose assay, and glucosamine was identified as the principal amino sugar in shell hydrolysates. In vivo labelling of the S. mansoni eggshell was demonstrated following injection of C14-glycine and C14-tyrosine into infected mice and subsequent purification of the shells of eggs recovered from their liver.

Species specificity of the immediate hypersensitivity to schistosomal proteolytic enzyme.

An acidic proteolytic enzyme which digests host haemoglobin can be isolated and purified from schistosomes. This small glycoprotein is an allergen which sensitizes the host, as shown by immediate hypersensitivity reactions. These are specific for either Schistosoma haematobium or S. mansoni and can be demonstrated by mast cell degranulation in mice or by intradermal skin tests in monkeys. Although high levels of total IgE may be found in acute and chronic schistosomiasis, there was no evident relationship between the worm burden in monkeys and immediate hypersensitivity reactions to either purified enzyme or crude schistosomal extracts. It is suggested that an in vivo correlation between worm burden and manifestations of the allergic response may be perturbed by high titres of non-specific IgE or other homocytotropic antibodies, thus accounting for false negative skin test reactions. Alternatively, a return to low or subnormal IgE levels may allow the restoration of the allergic response, giving rise to false positive reactions. Purified schistosomal antigens offer certain advantages over crude skin test preparations in terms of uniformity of antigen content, dosage and specificity. In addition, the enzyme may represent a species-specific tool for new immunochemical analyses of schistosomiasis.

Scanning electron microscope observations on tegument maturation in Schistosoma mansoni grown in permissive and non-permissive hosts.

Observations were made on details of tegument development of schistosomes grown in mouse, hamster, and rat hosts. In permissive hosts (mouse and hamster) the surface of the worm alters rapidly during early maturity and is characterized by fusing of a highly undulate surface network into smooth folds and spine-covered tubercles. In non-permissive hosts maturation of the tegument is both delayed and incomplete, and the tubercles are aspinous. Scanning views of the oral cavity and the gynegophoral canal, both sites of transitional tegumental organization, are also shown. The gynecophoral canal tegument seems to be a site of active lipid secretion.

Schistosoma mansoni tegumental appendages: scanning microscopy following thiocarbohydrazide-osmium preparation.

Scanning photographs of the surface of normal adult male and female Schistosoma mansoni are given. The specimens were prepared using a thiocarbohydrazide technique which facilitates the binding of osmium. The resultant deep penetration of osmium preserves surface detail for electron reflection. The schistosomal tegument is characterized by a variety of surface bosses and spines. The function of certain of these is thought to be tactile or as chemoreceptors. Numerous tegumental pores surrounding setae in surface tubercles are shown. Clefts in the inner aspect of the gynecophoral canal are also seen. The excretory pore in both male and female is shown, along with a cluster of receptors at the terminus of the gynecophoral groove. Such grouped receptors may function to indicate position of the worm in copula.

Influence of hycanthone on morphology and serotonin uptake of Schistosome mansoni.

The intrinsic content of serotonin (5HT) and the uptake of 5HT by Schistosoma mansoni taken from mice which were given a single intramuscular therapeutic dose of hycanthone were studied. Drug-exposed worms were found to have intrinsic values of 5HT which were similar or slightly less than controls. Uptake measurements were made on single and on paired worms recovered from mesenteric, portal, or hepatic sites and incubated in 75% horse serum or in Fischer's medium. All groups of treated worms were found to take up, on average, similar or lower amounts of 5HT compared to controls. These findings are in contrast to a recent report of very considerable increases in content or in 5HT acquisition in vitro by hycanthone-exposed parasites. This communication suggests that the mode of action of hycanthone cannot be explained as being due to increased 5HT uptake. Morphological changes in hycanthone-treated worms include loss of body weight and size, loss of hemoglobin pigment from the gut, deterioration of the tegument, and derangement of the vitellaria. The loss of gut contents occurs early after exposure to hycanthone and may indicate that interference with gut physiology and the nutritional state of the worms is one consequence of the drug, although the mechanism of these changes has not yet been elucidated.

Anticholinergic properties of the antischistosomal drug hycanthone.

The effect of the antischistosomal drug hycanthone on the motor activity of Schistosoma mansoni was studied in vitro. Hycanthone stimulates motor activity at concentrations of 10(-6) to 10(-5) M, and partially blocks the paralytic effects of carbachol and physostigmine. Lucanthone, a closely related although less active congener of hycanthone, does not produce these same effects in vitro. Some blocking of acetylcholine can also be produced by atropine, although this drug is less active in this regard than is hycanthone. These findings suggest that the therapeutic efficacy of hycanthone may be related to interference with acetylcholine receptors in schistosomes. Hycanthone is an inhibitor of acetylcholinesterase (ACHE) from S. mansoni, but is less effective against ACHE of mammalian origin. In contrast, physostigmine inhibits the mammalian enzyme more effectively than it does the helminth enzyme. These observations suggest that schistosome ACHE differs from the mammalian enzyme with respect to the configuration of the active center, and that hycanthone may have a selective affinity for schistosomal cholinergic systems.

Hypersensitivity to parasite proteolytic enzyme in schistosomiasis.

A proteolytic enzyme which hydrolyses hemoglobin was obtained from the supernatant fraction of homogenized Schistosoma mansoni. This enzyme elicited histaminic skin reactions in various animals, including man, which were infected with S. mansoni. It failed to induce reactions in monkeys harboring S. haematobium, S. japonicum, or S. intercalatum. In a preliminary field trial in the Caribbean, the skin test proved to be somewhat less sensitive than the customarily used extract of adult worms in Coca's solution. However, the enzyme appeared to induce fewer false positive reactions and delayed responses than did the Coca's extract. A new diagnostic test for schistosomiasis probably could be developed by using specific parasite enzymes against which the host has become sensitized in the course of infection.