Schistosoma mansoni: characterization of hemolytic activity from adult worms

Homogenates of adult Schistosoma mansoni worms contain a hemolytically active component(s). Centrifugation at 10,000 g shows the major activity is present in the pellet fraction. Red blood cell lysis with the schistosome hemolytic agent is optimal at acid pH (5.0) and highly temperature dependent. The hemolytic component is resistant to boiling (5 min) and stable for extended periods of time at 38 C (22 hr). The length of the lag phase prior to hemolysis and the rate of hemolysis are both concentration and temperature dependent. Following hemolysis, red blood cell ghosts remain.     

Schistosoma mansoni: pairing of male worms with artificial surrogate females

To determine whether male worms provide any specific polypeptides to females, we produced extruded alginate fibers that resembled the size and shape of mature female worms. Males clasped these surrogate "female worms" and remained "paired" with them for long periods. On polyacrylamide gel electrophoresis, fibers clasped for several days showed bands at approximately 40 and 46 kDa which were never found in fibers incubated in the same medium but not clasped by males. We believe that these may be substances transferred from male worms during normal pairing. Males biosynthetically labeled with [14C]leucine were permitted to pair with fibers, which took up a broad range of polypeptides visualized on long autoradiographic exposure to gels.     

Schistosoma mansoni: effects of antimony on immature and adult worms.

Immature Schistosoma mansoni in mice are less susceptible to antimony therapy than adult worms. KSb tartrate inhibited phosphofructokinase (PFK) (EC 2.7.1.11) to a greater extent in extracts of 3-week-old worms than adults, and inhibited production of lactate in both immature and adult worms in vitro. In vivo, KSb tartrate was accumulated similarly by 3-week-old worms and by adults: measurements of hexosephosphate following drug treatment suggested similar inhibition of PFK in the two worm stages. If antimony acts by inhibition of PFK it is not clear why the young worms are more resistant to chemotherapy than adults.     

Schistosoma mansoni: serotonin uptake and its drug inhibition

5-Hydroxytryptamine (serotonin) uptake by male and female Schistosoma mansoni was studied in vitro. Initial uptake was determined during the first 2 min of incubation with the indoleamine. There were two types of uptake: a saturable component that is apparent at low concentrations of serotonin, and a passive component that predominates at high concentrations. Female S. mansoni take up more 5-hydroxytryptamine per milligram protein than do the males. Studies on the distribution of 5-hydroxytryptamine taken up by the parasites show that a large proportion of the indoleamine is in the tegument. The female tegument had a greater proportion of the 5-hydroxytryptamine taken up than did the male tegument. The evidence indicated that 5-hydroxytryptamine associated with tegument represented the passive uptake while uptake in the main parasite body was both active and passive. Compounds that were shown to inhibit 5-hydroxytryptamine uptake include methylclonazepam, metergoline, imipramine, and ouabain. Maximal inhibition by these compounds ranged from 50 to 60%. Inhibition appeared to be confined to the saturable uptake component of the body fraction.     

Effect of immature Schistosoma mansoni worms on hamsters' plasma enzymes

1. The pattern and activity of isocitrate, lactate and malate dehydrogenases and malic enzyme were studied in plasma of normal hamsters and hamsters at the 26th day of infection with S. mansoni. 2. Although the electrophoretic patterns of these enzymes were similar in normal and infected hamsters, their activities were higher in the latter than the former group of animals. The elevation in the enzymic activity indicates that there is tissue damage caused by the larvae at this stage.     

Development of Schistosoma mansoni worms in mice analyzed by bright field and confocal microscopy

The blood flukes of mammals (Digenea: Schistosomatidae) are among trematodes unique whose adult worms have separated sexes which are dissimilar in appearance. The developmental features, growth and organogenesis of Schistosoma mansoni were studied in Swiss Webster mice by a digital system for image analysis and confocal microscopy. Data so far obtained showed two phases with significative morphological changes at 3-4 weeks post-infection, and a gradual similar development onwards in the reproductive system and tegument. Our male-dependent phase demonstrated that mating occurs before sexual maturing. At week three, the majority of male worms (59%) had formed the gynaecophoric canal although testicular lobes and tegumental tubercles were absent. By this time, 33% females had an incipient ovary (without cellular differentiation). At week four, 77.2% males presented testicular lobes with few germinative cells while 26% had developing tegumental tubercles. The immature ovary was observed in 69% females. Suckers followed different pattern of growth between male and females. The size of oral and ventral suckers from six-week-old male worms grew abruptly (3.0 fold) more than that of three-week-old. In female worms, maximum growth was attained at week four, reducing in size thereafter. From sixth week onwards, all specimens showed the fully developed reproductive system. Probably, these features are morphological traits which schistosome has experienced from hermaphrodite to dioecy.     

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.     

Transfer of [14C] cholesterol and its metabolites between adult male and female worms of Schistosoma mansoni

Adult male and female worms of Schistosoma mansoni are able to incorporate [14C]cholesterol and convert it into several metabolites. Schistosomula on the other hand cannot convert the incorporated cholesterol. Male worms are able to transfer the [14C]cholesterol and labelled metabolites to female worms but labelled female worms can transfer cholesterol but are unable to transfer the conversion products of cholesterol to male worms. Uptake by female worms of labelled products shed by male worms seems to occur more efficiently in the presence of serum than in its absence.     

Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized within the tegument of fixed worms on the cytoplasmic leaflet of both the double surface membrane and the basement membrane; this reaction was inhibited by 1 microM chlorpromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosomes are discussed.     

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.     

Schistosoma mansoni: uptake of exogenous hemeproteins by schistosomules grown in vitro

The uptake and fate of the hemeproteins horseradish peroxidase (HRPO) and hemoglobin (Hb) by schistosomules of Schistosoma mansoni maintained in vitro were studied by electron microscopy and cytochemical techniques. After administration of HRPO, reaction product was observed initially in the lumen of the digestive tract, and, after 2 hr of feeding, reaction product was also visible in the cytoplasm of the gastrodermis. There was no evidence of pinocytosis. After administration of Hb, reaction product was observed only in the lumen of the digestive tract. As is found following red blood cell feeding, digestive pigment was formed in the lumen of the gut following Hb feeding. The possible significance of these findings is discussed.     

Schistosoma mansoni: activation of hemolytic activity in homogenates from live adult worms

We have previously described hemolytic activity in extracts of lyophilized Schistosoma mansoni adult worms. In contrast, freshly homogenized live worms have little hemolytic activity. However, preincubation of the homogenate at 38 C, pH 5.1 for 22 hr resulted in an 18 fold increase in specific activity (Hb released/mg protein) due to dramatic increases in hemolytic activity and decreases in protein concentration. No activation occurred when the homogenate was boiled prior to preincubation or when preincubation was performed at 4 C. In addition, a thermostable inhibitor of hemolytic activity is present in freshly homogenized live adult S. mansoni. Hydrolysis of the inhibitor and activation of the hemolytic agent appear to be due in part to hydrolytic activity of one or more cysteinyl proteinases.     

Schistosoma mansoni: adhesion of mannan-binding lectin to surface glycoproteins of cercariae and adult worms

Schistosoma mansoni is a blood-dwelling trematode which can persist for several years in the vessels of the human host. The schistosomal surface has been extensively characterized by lectin binding studies, revealing the carbohydrate composition of the worm's tegument. Using fluorescent and scanning electron microscopy we demonstrate that the surface carbohydrates of cercariae and adult worms are the binding ligands for mannanbinding lectin (MBL), a serum protein that is part of the innate immune system. An in vitro complement activation assay with C1q-deficient complement suggests that MBL, in association with the serine proteases MASP-1 and MASP-2, is capable of fixing complement components on the schistosomal tegument and activating the complement cascade via the "MBL pathway." MBL is constitutively expressed by hepatocytes and present in the blood at a stable level. Since it is also a weak acute-phase protein and therefore upregulated in an acute-phase response we investigated the serum MBL levels in patients infected with Schistosoma sp. and in healthy control persons. An enzyme-linked immunosorbent assay indicated no differences between the two groups. Although our results suggest an involvement of MBL activated complement in vitro, its role in vivo remains to be clarified.     

Methionine uptake of larval and adult Schistosoma mansoni;

The uptake of methyl-¹⁴C-methionine by schistosomula, 21 day-old and adult Schistosoma mansoni in vitro has been investigated. Ligation of the adult pharynx and comparison between the kinetics of uptake of schistosomula and adults suggests that methionine is absorbed, in the system described, primarily via the tegument. All stages of the parasite examined absorb methionine by, at least, two kinetically distinguishable systems, one which is saturable and a second which appears to be simple diffusion. The saturable system has relatively low specificity for amino acids but shows no affinity for other classes of compounds. The effects of pH, temperature, metabolic inhibitors, and sodium ion concentration have been examined. The results are discussed with reference to schistosome gut function and also to the differential response to chemotherapy according to age of infection.     

The role of Schistosoma mansoni males in feeding and development of female worms

Female Schistosoma mansoni from unisexual infections have scant pharyngeal musculature, thin intestinal cecal walls, pale and scanty intestinal contents, and lack acidic thiol proteinase digestive enzyme as determined by indirect immunofluorescence using a monoclonal antibody. Their intake of host erythrocytes, measured by 51Cr labeling, is about one-fourth that of paired adult females, and they appear to be starved. In contrast, paired adult females have heavier pharyngeal musculature and intestinal cecal walls and abundant digestive enzyme in the anterior third of their intestinal tract. Females in worm pairs surgically transplanted into uninfected mice continued to feed, but separated females were carried into the liver and deteriorated. Adult female S. mansoni, newly separated from their male partners and incubated in vitro with labeled erythrocytes, ingested marginally fewer cells than did still-paired females, indicating their ability to continue feeding almost normally at least for a period after separation. Paired and ex-paired adult females declined similarly in feeding rate with increased time in vitro. In Schistosomatium douthitti, females grow and mature without males, the pharyngeal musculature and cecal walls are well developed, the gut is full of ingested blood, and the acidic thiol proteinase is present in both unisexual and paired female worms. There are different stimulatory pathways for growth and for reproductive maturation in S. mansoni, although both processes require physical contact with the male. We believe that the growth-stimulating function results from the muscular action of the clasping male, which helps the immature female to pump blood into her intestine, thereby overcoming a state of relative starvation.     

Morphometric study of Schistosoma mansoni adult worms recovered from Undernourished infected mice

Some unfavourable effects of malnutrition of the host on Schistosoma mansoni worm biology and structure have been reported based upon brigthfield microscopy. This paper aims to study by morphometric techniques, some morphological parameters in male and female adult worms recovered from undernourished albino mice in comparison with parasites recovered from well-fed infected mice. Undernourished animals were fed a multideficient and essentially low protein diet (RBD diet) and compared to well-fed control mice fed with the commercial diet NUVILAB. Seventy-five days post-infection with 80 cercarie (BL strain) animals were sacrificed. All adult worms were fixed in 10% formalin and stained with carmine chloride. One hundred male and 60 female specimens from each group (undernourished and control) were examined using an image system analysis Leica Quantimet 500C and the Sigma Scan Measurement System. The following morphometrical parameters were studied: body length and width, oral and ventral suckers, number and area of testicular lobes, length and width of ovary and uterine egg. For statistical analysis, the Student's t test for unpaired samples was applied. Significant differences (p < 0.05) were detected in body length and width, in parameters of suckers, uterine egg width, ovary length and area of testicular lobes, with lower values for specimens from undernourished mice. The nutritional status of the host has negative influence on S. mansoni adult worms, probably through unavailability of essential nutrients to the parasites.