Schistosoma mansoni: effects of in vitro serotonin (5-HT) on aerobic and anaerobic carbohydrate metabolism

The effect of Serotonin on carbohydrate metabolism, excreted end products, and adenine nucleotide pools in Schistosoma mansoni was determined following 60 min in vitro incubations under air (= 21% O2) and anaerobic (95% N2:5% CO2) conditions. In the presence of 0.25 mM Serotonin, glucose uptake increased by 82-84% and lactate excretion increased by 77-78%; levels of excreted lactate were significantly higher under aerobic than under anaerobic conditions. The tissue pools of glucose, hexosephosphates, fructose 1,6-bisphosphate, pyruvate, and lactate were significantly increased under anaerobic conditions compared to air incubation; the presence of Serotonin decreased tissue glucose pools and increased the size of the pyruvate and lactate tissue pools. The glycolytic carbon pool was significantly greater under anaerobic than under aerobic conditions, irrespective of Serotonin. Serotonin increased adenosine 5'-diphosphate and adenosine 5'-monophosphate levels under aerobic conditions; neither Serotonin nor gas phase significantly affected total adenine nucleotide levels or the adenylate energy charge. Serotonin increased energy requirements by S. mansoni due to increased muscle contractions; demand was met by enhanced rates of carbohydrate metabolism. Irrespective of gas phase, 74-78% of available carbohydrate was converted to lactate. In the presence of Serotonin, conversion of glucose to lactate was reduced to 63-67%. In view of the requirements by S. mansoni for an abundant supply of glycoprotein and glycolipid precursors for surface membrane renewal, it is suggested that carbohydrate (glucose and glycogen) that was not converted to lactate may have been incorporated into biosynthetic processes leading to membrane synthesis.     

In vitro effects of artesunate on the survival of worm pairs and egg production of Schistosoma mansoni

The effect of artesunate (ART) on the survival time of adult worm pairs of Schistosoma mansoni and on their egg output during in vitro culture was assessed. ART significantly decreased the survival time of both paired male and female worms at concentrations of 5, 10, 20 and 40 mg l- 1 during in vitro cultivation. An inhibitory effect of ART on the daily egg output of paired female worms during in vitro cultivation was also observed.     

Sugar transport and metabolism in Schistosoma mansoni.

The absorption kinetics of some 14-C-labeled simple sugards in adults of Schistosoma mansoni are described. The influx of fructose and 3-0-methylglucose was by diffusion alone, while glucose, 2-deoxyglucose (2DOG), galactose, glucosamine, and mannose were absorbed by mediated transport as well as by diffusion. Although absorbed glucose was rapidly metabolized, uptake rates of radio-glucose in 2-min incubations corresponded with the amount of glucose (determined chemically) removed from the incubation medium. In 30-min incubations 2DOG was slowly metabolized and accumulated against an apparent concentration difference. The mediated transport of glucose and 2DOG was inhibited in Na+-free media, and by the presence of ouabain, phlorizin, phloretin, and other sugars. Accordingly, influxes of glucose of 2DOG and 22-Na+ were coupled. On a per mg protein basis, female worms transported more 2DOG and glucose, but less glycine, than did males. However, the rate of glucose metabolism by male and female worms incubated together was greater than that of either males or females incubated separately. The nature of sugar transport in schistosomes and other flatworms is similar to that in vertebrates.     

Herbimycin A suppresses mitotic activity and egg production of female Schistosoma mansoni

The eggs of the endoparasite Schistosoma are the causative agent of schistosomiasis, an important disease of humans, which is endemic in (sub-) tropical regions. The absence of a vaccine with sufficient protective qualities and increasing resistance to approved and established drugs like praziquantel, justify the exploration of novel ways to fight schistosomes. Our strategy is based on interference with the sexual maturation of the female. Prerequisites for gonad development in adult females are a continuous pairing contact with the male and significantly increased mitotic activity. In this study we show that the male governs sexual maturation of the female, as the separation of couples causes a clear reduction of female mitotic activity and, consequently, egg production. We demonstrate that treatment of schistosomes with Herbimycin A, an inhibitor of protein tyrosine kinases (PTKs), mimics the separation of couples as the drug blocks mitotic activity and egg production of paired females. However, the synthesis of the eggshell precursor protein p14 is elevated. Furthermore, we show for the first time in invertebrates that Herbimycin A decreases tyrosine phosphorylation and PTK stability in schistosomes. Summarised, our data provide evidence that PTKs have key functions in regulating gonad development, eggshell gene expression and, consequently, egg production. Therefore, we suggest envisaging schistosome PTKs as novel targets for strategies to combat schistosomiasis.     

Schistosoma mansoni: egg morphology and hatchability

Schistosoma mansoni eggs were isolated and staged microscopically according to size. The eggs were then allowed to hatch and were reexamined; the hatching percentage of mature eggs was calculated. The results show clearly that not only do immature eggs not hatch, but also mature eggs greater than 160 microns fail to hatch.     

Schistosoma mansoni: incorporation and metabolism of protein amino acids in vitro

1. The amino acid composition of total proteins in six stages of the life cycle of Schistosoma mansoni was determined by routine autoanalysis of acid hydrolysates. Aspartate, glutamate and glycine were consistently the most abundant protein amino acids in all stages. 2. Incorporation of each of the protein amino acids into adult and egg proteins was determined using 72 hr cultures in complex media. Incorporation rates varied widely and there was no correlation between abundance in protein and the rate of incorporation. 3. Only five amino acids were interconverted to other amino acids which were themselves incorporated into worm and egg proteins (ala, arg, asp, gly, ser); of these only two (glu from ala and pro from arg) appeared to be of quantitative significance. Exogenous glucose yielded only three protein amino acids (ala, asp, glu). 4. The data are considered in the light of differences in egg and adult protein synthesis and with particular regard to potential chemotherapy at this level.     

Acidic thiol proteinase activity of Schistosoma mansoni egg extracts.

Extracts of the eggs of the human blood fluke, Schistosoma mansoni, exhibit proteolytic activity which requires the presence of added thiol reagents or cyanide. The pH optimum for hydrolysis of Azocoll and cartilage proteoglycan is 4.8--5.2 and the molecular weight of the major component is 25--26,000. The effects of inhibitors suggest this activity belongs to the acidic thiol proteinase class, with a similarity to Cathepsin B. These proteinases may be involved in nutrition of the egg or sporocyst, in penetration of eggs or miracidia through host tissues, or in the immunopathology of schistosomiasis.     

Enzyme activities in Schistosoma mansoni soluble egg antigen

Schistosoma mansoni egg antigens are mostly responsible for the granulomatous pathology in human intestinal schistosomiasis. Several previous studies have indicated that the induction of an immune response against some parasite enzymes may protect against pathology. The present work was designed to identify enzyme activities present in a standard soluble egg antigen (SEA) preparation. Simple colorimetric analyses were performed incubating SEA with 2-naphthyl, 2-naphthylamide (2NA), or p-nitrophenyl substrates at different pHs in the absence of added effectors. Results showed prominent acid phosphatase (pH 5.4), alkaline phosphatase (pH 8.5), and N-acetyl-beta-glucosaminidase (pH 5.4) activities. Relevant peptidase activities were also detected at pH 6.5-7.5 against 2NA derivatives of (1) aliphatic (alpha-Ala > beta-Ala > Leu > Met > S-benzyl-Cys), polar (Ser > Gln), basic (Arg > Lys > ornithine), and acidic (Glu) amino acids; (2) dipeptides: X-Ala (X = Gly > Leu > Lys > Asp), X-Arg (X = Ala > Arg > Phe > Gly > Pro > Asp), Ser-Met, and Phe-Pro; and (3) tripeptides (Ala-Phe-Pro > Phe-Pro-Ala). The data demonstrated that S. mansoni SEA contains a rich set of hydrolases with different specificities that might play a role in the egg physiology and possibly also in the host-parasite relationships.     

Glycomics analysis of Schistosoma mansoni egg and cercarial secretions

The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries. Glycoconjugates, and in particular the carbohydrate component of these products, represent the main immunogenic challenge to the host and could therefore represent one of the crucial determinants for successful parasite establishment. Here we report a comparative glycomics analysis of the N- and O-glycans derived from glycoproteins present in S. mansoni egg (egg-secreted protein) and cercarial (0-3-h released protein) secretions by a combination of mass spectrometric techniques. Our results show that S. mansoni secrete glycoproteins with glycosylation patterns that are complex and stage-specific. Cercarial stage secretions were dominated by N-glycans that were core-xylosylated, whereas N-glycans from egg secretions were predominantly core-difucosylated. O-Glycan core structures from cercarial secretions primarily consisted of the core sequence Galbeta1-->3(Galbeta1-->6)GalNAc, whereas egg-secreted O-glycans carried the mucin-type core 1 (Galbeta1-->3GalNAc) and 2 (Galbeta1-->3(GlcNAcbeta1-->6)GalNAc) structures. Additionally we identified a novel O-glycan core in both secretions in which a Gal residue is linked to the protein. Terminal structures of N- and O-glycans contained high levels of fucose and include stage-specific structures. These glycan structures identified in S. mansoni secretions are potentially antigenic motifs and ligands for carbohydrate-binding proteins of the host immune system.     

Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.     

Genetic knockdown and pharmacological inhibition of parasite multidrug resistance transporters disrupts egg production in Schistosoma mansoni

P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) are ATP-dependent transporters involved in efflux of toxins and xenobiotics from cells. When overexpressed, these transporters can mediate multidrug resistance (MDR) in mammalian cells, and changes in Pgp expression and sequence are associated with drug resistance in helminths. In addition to the role they play in drug efflux, MDR transporters are essential components of normal cellular physiology, and targeting them may prove a useful strategy for development of new therapeutics or of compounds that enhance the efficacy of current anthelmintics. We previously showed that expression of Schistosoma mansoni MDR transporters increases in response to praziquantel (PZQ), the current drug of choice against schistosomiasis, and that reduced PZQ sensitivity correlates with higher levels of these parasite transporters. We have also shown that PZQ inhibits transport by SMDR2, a Pgp orthologue from S. mansoni, and that PZQ is a likely substrate of SMDR2. Here, we examine the physiological roles of SMDR2 and SmMRP1 (the S. mansoni orthologue of MRP1) in S. mansoni adults, using RNAi to knock down expression, and pharmacological agents to inhibit transporter function. We find that both types of treatments disrupt parasite egg deposition by worms in culture. Furthermore, administration of different MDR inhibitors to S. mansoni-infected mice results in a reduction in egg burden in host liver. These schistosome MDR transporters therefore appear to play essential roles in parasite egg production, and can be targeted genetically and pharmacologically. Since eggs are responsible for the major pathophysiological consequences of schistosomiasis, and since they are also the agents for transmission of the disease, these results suggest a potential strategy for reducing disease pathology and spread.     

Aerobic and anaerobic metabolism in Entamoeba histolytica

Respiration by Entamoeba histolylica is confirmed. A doubling of the rate of oxygen uptake was observed upon the addition of d-glucose to cells in which the glycogen reserve had been partially depleted. In cells metabolizing endogenous substrates the rate of oxygen uptake was not influenced by sodium cyanide or sodium succinate. It was slightly depressed when d-mannose was the added sugar. The end products, CO₂, ethanol, and acetate accounted for essentially all of the glucose carbon utilized in both aerobic and anaerobic experiments. The radioactivity from uniformly labelled ¹⁴C-glucose was found in these products. Three times as much ethanol as acetate was produced in the anaerobic experiments and in the aerobic experiments this ratio was approximately reversed.     

Aerobic and anaerobic metabolism during activity in small rodents.

Analysis of oxygen consumption and lactic acid formation during five minutes of maximal activity by the rodents Microtus montanus (Cricetidae) and Dipodomys merriami (Hetermyidae) indicates that: (1) anaerobiosis provides approximately 10% of total energy utilized during the 5-minute activity period; (2) anaerobiosis may account for as much as one-third of total energy utilized during the first 30 seconds of activity. In addition, these data indicate at least one species of lizard may be capable of a higher total rate of metabolism during "burst" activity than are the rodents investigated here.