Schistosoma mansoni: partial purification and properties of ornithine-delta-transaminase.

Ornithine-δ-transaminase (OTA) (EC 2.6.1.13) was isolated from Schistosoma mansoni and purified more than 16-fold. Treatment of the worm homogenate with 0.4% deoxycholate (DOC) in the presence of 0.8 M KC1 and 0.15 M NaCl at pH 8.3 resulted in solubilization of 85% of the enzyme. Sonication and high-speed centrifugation were unnecessary. The solubilization procedure and the subsequent purification steps required the presence of the coenzyme pyridoxal phosphate. The optimal pH for OTA was 8.5 and the optimal incubation temperature was 55 C. Michaelis-Menten constants (K_m) for ornithine and α-ketoglutarate were 1.53 mM and 2.07 mM, respectively, in enzyme preparations with a specific activity of 22–29 μmoles/hr/mg protein. The enzyme showed a high affinity for α-ketoglutarate but considerably less affinity for oxaloacetate and pyruvate. High concentrations of α-ketoglutarate and ornithine inhibited the OTA activity. Similarly inhibitory were the structurally related amino acids isoleucine and serine and also oxaloacetate. The K_m for α-ketoglutarate in the presence of oxaloacetate was 1.3 mM and the V_max was 8.38 μmoles/hr/mg protein.     

Schistosoma mansoni: glycosyl transferase activity and the carbohydrate composition of the tegument.

Homogenates of adult Schistosoma mansoni contain enzymes which transferred [¹⁴C]mannose, [¹⁴C]glucose, and [¹⁴C]galactose from GDP-[U-¹⁴C]mannose, UDP-[U-¹⁴C]glucose, and UDP-[U-¹⁴C]galactose respectively to a lipid acceptor; in comparison, free [¹⁴C]mannose, GDP-[U-¹⁴C]fucose, and UDP-[U-¹⁴C]acetyl-glucosamine were poorly transferred. The lipid acceptor is believed to be an intermediate in the glycosylation of the worm's glycoproteins and in the biosynthesis of oligosaccharides and glycolipids. The tegument of adult worms was isolated by the freeze-thaw procedure and sugars associated with macromolecules in this fraction were analyzed; the major monosaccharide components were glucose, galactose, and mannose. These results suggest that the mechanism of glycosylation of the adult schistosome's tegumental macromolecules may occur through the glycosyl transferase system. The schistosome mannosyl transferase (EC 2.4.1), which is membrane bound was solubilized with 0.1% Triton X-100 without loss of activity; after density gradient centrifugation there was a peak of enzymic activity in a region of density 1.08, which could not be associated with any particular organelle.     

Schistosoma mansoni: neurotransmitters, longitudinal musculature and effects of electrical stimulation

Longitudinal muscle shortening of adult male Schistosoma mansoni is produced by electrical stimulation. Responses are frequency and strength dependent. Neither 5-hydroxytryptamine (5-HT) antagonists nor dopamine interfere with the response. 5-HT does not enhance it. Carbachol (10^?4M), eserine (10^?6M), and metrifonate (10^?5M) block the response but acetylcholine alone has no affect. Atropine (10^?4M) partially counteracts the effects of carbachol. Hyperosmotic sucrose but not urea blocks the stimulation response. It is concluded that the response is nonneuronally conducted via a pathway involving gap junctions and that neurotransmitters probably act as modulators of motor activity rather than as initiators of it.     

Schistosoma mansoni: physical and chemical factors affecting the mechanical properties of the adult male musculature.

The response of the musculature of male Schistosoma mansoni to various physical and ionic environments was determined. Contractile activity and tension of the parasite's musculature decreased when they were incubated in buffered salt solutions having an osmolality greater than 300 mOsm, a pH less than 6.8, or a temperature greater than 39 C. Except for potassium, high concentrations of inorganic ions reduced the tension of the parasite's musculature; high concentrations of potassium increased tension. In general the contraction rate of the male schistosome decreased when the concentration of an inorganic ion was below or above that found in Hanks' balanced salts solution. These results indicate that the musculature of S. mansoni is similar to smooth muscle found in mammals.     

Schistosoma mansoni: early antimonial treatment of infected mice

The main effect of antimonial treatment in the early phases of schistosome infection is due to an interference with the development of the worm. This effect manifests itself in two different forms: one is a temporary (reversible) delay of development and/or growth, the other, an irreversible blocking of development, leading to the reduction of worm recovery. The antimonials, besides their lethal and toxic effects on the adult worm, exert in vivo a “schistosomistatic” action of variable intensity and duration. The earlier the treatment, the more pronounced is this action, reaching its maximum at the time of cercarial exposure. As a consequence of the temporary delay of the development, the number of the worms became higher in the autopsies conducted at longer intervals from the cessation of treatment. The delay in growth in some cases was followed by a lethal action.     

Schistosoma mansoni: a complement-dependent receptor on adult male parasites

The dorsal surface of adult male Schistosoma mansoni exhibits an affinity for Salmonella typhi when these bacteria are preincubated in normal serum from mice, guinea pigs, or humans. The complement (C) system was shown to be responsible for the bacterial binding. Bacteria not preincubated with normal serum or preincubated in normal serum which had been treated with ethylenediamine tetraacetic acid (EDTA), or cobra venom factor (CoF), or heated at 56 C for 60 min did not bind to the parasite's surface. Further experiments utilizing ethylene glycol tetraacetic acid (EGTA) plus Mg²⁺, heat inactivation at 50 C for 30 min, and zymosan treatment of the serum indicated the C fixation and deposition on the bacteria occurs via the alternative C pathway. These observations indicate the presence of a complement-dependent receptor on the dorsal tegumental surface of the adult male parasite.     

Schistosoma mansoni: adult worm chemoattraction with barriers of specific molecular weight exclusions

Chemoattraction studies were done with Schistosoma mansoni adults from mice. To test for attraction or repulsion, some worm pairs were separated mechanically and the individuals placed in polycarbonate chambers. Experiments were done at 37 C and chambers contained dialysis tube chimneys. In all cases, heterosexual attraction occurred when one worm, but not two worms, were placed in the chimneys. Unperforated chimneys with specific molecular weight (Mr) exclusions were compared with perforated chimneys to study heterosexual attraction. Attraction was similar in designs using perforated chimneys and those with 50,000 and 12,000 Mr exclusions, but none was seen in chimney designs with 1000 Mr exclusions.     

Schistosoma mansoni: cryopreservation of schistosomules.

Conditions were established for recovery of active schistosomules of Schistosoma mansoni after cryopreservation and storage in liquid nitrogen (?196 C). Schistosomules prepared from cercariae by a shear pressure technique were subjected to a two-step cooling process consisting of a slow cooling rate to an intermediate temperature, followed by rapid quenching of the sample in liquid nitrogen. Overall averages of 39 and 44% of the schistosomules, with a maximum of 88%, were recovered retaining normal activity with cooling rates of 0.4 C/min to ?32 C or 0.8 C/min to ?35 C, respectively. Methanol at 17.5% in Earle's lactalbumin hydrolysate was the freezing medium. As compared with 24 hr storage in liquid nitrogen, no loss in schistosomule motility was observed after 1 month. Following cryopreservation, attenuated schistosomules derived from ⁶⁰Co-irradiated cercariae (50 kR) exhibited structure and activity equivalent to that of unattenuated schistosomules. Infectivity for mice of unattenuated schistosomules derived from ⁶⁰Co-irradiated cercariae (50 Krad) exhibited structure and activity of unfrozen schistosomules ranged from 0.4 to 15.2%.     

Schistosoma mansoni: patter of release of secretory products.

Adult Schistosoma mansoni were maintained in vitro for 1 hr with radioactively labeled precursors of protein, glycoprotein, and polysaccharides. The worms were then washed extensively and the supernates analyzed. The precursors N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, leucine, and fucose were incorporated into the worms and both large and small molecular weight products accumulated in the supernatant. For all the precursors except fucose, there was an initial rapid and then slower phase of release for both the large and small molecular weight materials. The amount of label retained by the worms as well as the proportion excreted as large molecular weight material was characteristic for the precursor used. In contrast, the products of fucose were released within 4 to 6 hr and therefore only exhibited the early secretory phase. There was no retention of fucose by the worms. Hydrolysis of large molecular weight products revealed that the N-acetylglucosamine-derived material was incorporated as amino sugars and fucose was incorporated as fucose. Therefore, N-acetylglucosamine and fucose precursors can specifically label secretory glycoproteins of schistosomes in a manner similar to that in mammalian systems.     

Schistosoma mansoni: Mechanisms in regulation of glycolysis

Adult pairs of Schistosoma mansoni convert glucose to lactate rapidly and almost quantitatively under aerobic and anaerobic conditions E. Bueding, 1950, Journal of General Physiology33, 475–495). Glycolysis is the principal source of energy of schistosomes and its inhibition by trivalent organic antimonials, at the phosphofructokinase step [EC 2.7.1.11], may be the basis for the chemotherapeutic effects of these agents E. Bueding and J. M. Mansour, 1957, British Journal of Pharmacology and Chemotherapy12, 159–165). We have developed standardized conditions for the comparison of rates of glucose consumption and lactate production by intact schistosomes in vitro and by centrifuged homogenates of worms. The rates of glycolysis of homogenates prepared from freshly isolated worms, and from worms that have been lyophilized immediately after harvesting and stored for prolonged periods at ?80 C were identical, when measured in media containing appropriate concentrations of glucose, NAD, ATP, MgCl₂, KCl, and phosphate. The specific activities of the 11 glycolytic enzymes and of 3 related enzymes (fructose-biphosphatase [EC 3.1.3.11], glycerol-3-phosphate dehydrogenase [EC 1.1.1.8], and malate dehydrogenase [EC 1.1.1.37]) were measured in homogenates under optimal conditions. The profile of the relative activities of glycolytic enzymes of S. mansoni resembles closely that of Ehrlich ascites tumor cells, and differs markedly from that observed in erythrocytes or skeletal muscle. As is the case in many animal tissues, hexokinase [EC 2.7.1.1] was the enzyme of lowest specific activity, and the rate of glycolysis of homogenates was almost the same as the hexokinase activity. Several other lines of evidence support the view that the hexokinase reaction is the rate-limiting step in the glycolysis of worm homogenates. Hexokinase activity was not particulate in schistosome homogenates, and there was no detectable high K_m glucokinase-like activity. The rate of glycolysis by homogenates exceeded that of intact worms by a factor of nearly 5. The contributions of glucose transport, availability of ADP and inorganic phosphate, regulatory enzymes, and a substrate cycle catalyzed by fructose-bisphosphatase are considered as possible mechanisms for the restraint of glycolysis in intact worms. The mechanisms contributing to the rapid rates of glycolysis of adult S. mansoni have not been identified, although several can be excluded (unusually high capacity of the glycolytic enzymes, the presence of mitochondrial hexokinase, the occurrence of glycosomes, and the operation of defective mitochondrial shuttles). In view of the regulatory role of hexokinase in the glycolysis of S. mansoni, inhibition of this enzyme is a potentially important target for the development of new antischistosomal drugs.     

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: peripheral and tissue eosinophilia in infected rats.

Peripheral eosinophilia is induced in Sprague-Dawley rats following infection with cercariae of Schistosoma mansoni. Beginning 3 weeks after infection, peripheral eosinophil levels rise above the baseline range; they reach peak values during the fifth week. Following a decline from peak values, peripheral eosinophil levels remain elevated and are observed to fluctuate for the next 5 months. The magnitudes of both the initial peak response at 5 weeks and the subsequent chronic level of peripheral eosinophils are dependent upon dose of cercariae. The initial peak response phase of peripheral eosinophilia coincides in time with the period of adult worm elimination (Weeks 4–6) in the schistosome-infected rat. Histological examination of the liver at 5 weeks after infection reveals eosinophil-rich inflammatory reactions associated with both live and dead worms residing in the portal blood vessels. Around live worms the inflammatory cells are localized in a perivascular arrangement; around dead worms these cells are in the vascular lumen in contact with destroyed worms. The chronic phase of peripheral eosinophilia is associated, in part, with inflammatory reactions surrounding eggs deposited in the liver by the few worm pairs which survive more than 6 weeks and remain within the liver. Histological examination during this period reveals granulomatous lesions within the liver surrounding eggs and dead worms. The granulomas are predominately monocytic (lymphocytes, macrophages) at 11 and 16 weeks. The initial peak response phase of peripheral eosinophilia appears to be a marker for tissue-localized reactions of eosinophils with worms. There are relationships between inflammatory reactions and survival of adult worms.     

Schistosoma mansoni: evaluation of an RNAi-based treatment targeting HGPRTase gene

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential gene of the parasite Schistosoma mansoni and it is well conserved in its hosts (mouse and human) at the protein but not at the RNA level. This feature prompted us to assess RNA interference (RNAi) to combat schistosomiasis. Small interfering RNAs (siRNAs) were produced against HGPRTase, injected in infected mice and the number of worms was counted six days after injection. The total number of parasites was reduced by approximately 27% after treatment. RT-PCR analyzes showed a significant reduction in parasite target mRNA but not in host's homologue. The use of low doses of molecules did not oversaturate si- or miRNA pathways as mice survival rates were not affected by siRNAs. This is the first successful in vivo demonstration of a RNAi-based treatment against schistosomiasis. We believe that improvements in molecule delivery and an increase on siRNA dose could rapidly eliminate parasite.     

Schistosoma mansoni: circulating antigens and immune complexes in infected mice.

Circulating schistosome antigens (CSA) and circulating immune complexes (CIC) were investigated during the course of Schistosoma mansoni infection in mice. The radioimmunoprecipitation-polyethylene glycol (PEG) assay (RIPEGA) with [¹²⁵I]anti-S. mansoni antibodies or [¹²⁵I] anti-antigen “4” antibodies detected, respectively, total CSA and antigen “4” in serum and in 3% polyethylene glycol-precipitated CIC from infected mice. Complement fixation test and [¹²⁵I] C_1q-binding test revealed, respectively, an anticomplementary activity and the presence of C_1q-binding CIC. All these substances appeared in infected mice at approximately the same period, i.e., between the 40th- and the 55th- day postinfection. No correlation was observed between the detection of anticomplementary active substances and C_1q-binding CIC. In contrast, a close relationship was noticed between CSA and complement-activating material during the course of the infection. This suggests that substances with anticomplementary activity in serum from infected mice could be one or various CSA. A close correlation was also observed between C_1q-binding CIC and free or “complexed” antigen “4.” This observation supports well the possibility that antigen “4” is one of the major complexed circulating antigen present in schistosomiasis. The immunoglobulins G₁, G_2a, M, and A were also characterized in 3% PEG-precipitated CIC from infected mice during the period in which we detected C_1q-binding CIC. The roles played by specific S. mansoni CIC in either schistosomal nephropathy or protective mechanisms to a challenge infection in mice are discussed.     

Schistosoma mansoni: complement and cercarial tail loss during in vitro transformation.

When Schistosoma mansoni cercariae are incubated at 37 C in media containing serum, the organisms lose their tails and change into viable, infective schistosomula. Tail loss does not occur in the absence of serum, or when the serum is heat inactivated. In the present studies, tail loss during in vitro conversion was shown to be complement dependent. The capacity of fresh serum to promote tail loss was markedly suppressed or abolished by cobra venom factor, zymosan, Sepharose CL-4B AND anti-C3 antibody. The alternative rather than the classic complement pathway appeared to be responsible since (1) binding of anti-C3 to cercariae required magnesium, but not calcium; (2) both C4-deficient serum and C2-deficient serum supported tail loss; but (3) human serum heated to 50 C for 20 min to inactivate Factor B did not support tail loss. Cercarial tail loss also required the terminal complement components C5 through C8. The extent and rate of tail loss was normal in agammaglobulinemic sera indicating that the complement effect was not antibody dependent.     

Schistosoma mansoni: thiol proteinase properties of adult worm "hemoglobinase".

This report presents evidence that the “hemoglobinase” from adult Schistosoma mansoni, first described by Timms and Bueding and later by Senft and his collaborators, belongs to the class of thiol proteinases. Proteolytic activity is stimulated by SH-containing compounds and inhibited by N-ethylmaleimide as well as other inhibitors of thiol proteinases. The enzyme can be partially purified by affinity chromatography using a Sepharose-linked organomercurial ligand. In addition to its activity on globin and hemoglobin, the enzyme can also be assayed with Azocoll, a general protease substrate, and by the activation of inactive trypsinogen to active trypsin. Extraction of the enzyme is enhanced by the addition of the nonionic detergent Triton X-100.     

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.     

Schistosoma mansoni: vaccination of mice with cryopreserved irradiated schistosomules.

In our earlier experiments, NIH/Nmri (CV) mice developed protective immunity to a Schistosoma mansoni cercarial challenge when previously exposed percutaneously to highly ⁶⁰Co-irradiated homologous cercariae. Experiments reported here were conducted to assess the immunogenicity of unfrozen and frozen and thawed schistosomules derived from ⁶⁰Co-irradiated cercariae (irradiated schistosomules). Immunization of NIH/Nmri (CV) mice by ⁶⁰Co-irradiated unfrozen schistosomules reduced worm burdens from a subsequent percutaneous challenge with normal cercariae by 41 to 72%. Immunogenicity was not narrowly dependent on irradiation dose rates between 1 and 8 kR/min, or on the total dose of irradiation given the schistosomules between 25 and 50 kR. Comparable protective immunity developed after injection of irradiated schistosomules which had been frozen to ?196 C in liquid nitrogen and thawed. Cryopreservation appears to offer a solution to the problem of storage of attenuated, immunogenic S. mansoni schistosomules.     

Schistosoma mansoni: stimulus and transformation of cercariae into schistosomules

Schistosoma mansoni schistosomules prepared from cercariae by seven in vitro techniques had not all reached the same state of development at the end of the incubation period as scored by seven parameters: water tolerance; Cercarienhüllen Reaktion; presence of the glycocalyx; condition of the surface membrane; nuclear state; granule migration; and cryopreservability. At the end of the specific incubation period for each technique, the level of development was judged with respect to schistosomules which had developed in situ for 1 hr after penetration of the ear skin of mice. In descending order of their correspondence to in vivo schistosomules, those derived in vitro (by the procedures listed) ranked as follows: first, penetration of dried rat skin; second, centrifuging and vortexing, or incubation in serum-supplemented medium; and third, syringe passage, omnimixing, centrifuging, and incubating, or incubating alone. The only treatment common to all techniques was incubation in 37 C culture medium for 2 hr or more. This is suggested as the stimulus for the cercaria-to-schistosomule transformation.