Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies.

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure-function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 µmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

Safety and efficacy of the rSh28GST urinary schistosomiasis vaccine: A phase 3 randomized, controlled trial in Senegalese children.

BACKGROUND: Urinary schistosomiasis, the result of infection by Schistosoma haematobium (Sh), remains a major global health concern. A schistosome vaccine could represent a breakthrough in schistosomiasis control strategies, which are presently based on treatment with praziquantel (PZQ). We report the safety and efficacy of the vaccine candidate recombinant 28-kDa glutathione S-transferase of Sh (rSh28GST) designated as Bilhvax, in a phase 3 trial conducted in Senegal. METHODS AND FINDINGS: After clearance of their ongoing schistosomiasis infection with two doses of PZQ, 250 children aged 6-9 years were randomized to receive three subcutaneous injections of either rSh28GST/Alhydrogel (Bilhvax group) or Alhydrogel alone (control group) at week 0 (W0), W4, and W8 and then a booster at W52 (one year after the first injection). PZQ treatment was given at W44, according to previous phase 2 results. The primary endpoint of the analysis was efficacy, evaluated as a delay of recurrence of urinary schistosomiasis, defined by a microhematuria associated with at least one living Sh egg in urine from baseline to W152. During the 152-week follow-up period, there was no difference between study arms in the incidence of serious adverse events. The median follow-up time for subjects without recurrence was 22.9 months for the Bilhvax group and 18.8 months for the control group (log-rank p = 0.27). At W152, 108 children had experienced at least one recurrence in the Bilhvax group versus 112 in the control group. Specific immunoglobulin (Ig)G1, IgG2, and IgG4, but not IgG3 or IgA titers, were increased in the vaccine group. CONCLUSIONS: While Bilhvax was immunogenic and well tolerated by infected children, a sufficient efficacy was not reached. The lack of effect may be the result of several factors, including interference by individual PZQ treatments administered each time a child was found infected, or the chosen vaccine-injection regimen favoring blocking IgG4 rather than protective IgG3 antibodies. These observations contrasting with results obtained in experimental models will help in the design of future trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT 00870649.

Polypyridylruthenium(II) complexes exert anti-schistosome activity and inhibit parasite acetylcholinesterases.

BACKGROUND: Schistosomiasis affects over 200 million people and there are concerns whether the current chemotherapeutic control strategy (periodic mass drug administration with praziquantel (PZQ)-the only licenced anti-schistosome compound) is sustainable, necessitating the development of new drugs. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the anti-schistosome efficacy of polypyridylruthenium(II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb12-tri and Rubb7-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb12-tri) and 10 mg/kg (Rubb7-tnl). Mice treated with Rubb12-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb12-tri-68%, Rubb7-tnl-56%) and were significantly morphologically altered (Rubb12-tri-62% abnormal, Rubb7-tnl-35% abnormal). We hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway. CONCLUSIONS/SIGNIFICANCE: Our data provide compelling evidence that ruthenium complexes are effective against all intra-mammalian stages of schistosomes, including schistosomula (refractory to PZQ) and eggs (agents of disease transmissibility). Further, the results of this study suggest that schistosome AChE is a target of ruthenium drugs, a finding that can inform modification of current compounds to identify analogues which are even more effective and selective against schistosomes.

Preparation and investigation of P28GST-loaded PLGA microparticles for immunomodulation of experimental colitis.

The aim of this work was to prepare and characterize (in vitro and in vivo) PLGA-based microparticles loaded with an enzymatic protein derived from the helminth parasite Schistosoma haematobium: glutathione S-transferase P28GST (P28GST). This protein is not only a promising candidate vaccine against schistosomiasis, it also exhibits interesting immunomodulating effects, which can be helpful for the regulation of inflammatory diseases. Helminths express a regulatory role on intestinal inflammation, and immunization by P28GST has recently been shown to be as efficient as infection to reduce inflammation in a murine colitis model. As an alternative to the combination with a classical adjuvant, long acting P28GST microparticles were prepared in order to induce colitis prevention. PLGA was used as biodegradable and biocompatible matrix former, and a W/O/W emulsion/solvent extraction technique applied to prepare different types of microparticles. The effects of key formulation and processing parameters (e.g., the polymer molecular weight, drug loading, W/O/W phase volumes and stirring rates of the primary/secondary emulsions) on the systems' performance were studied. Microparticles providing about constant P28GST release during several weeks were selected and their effects in an experimental model of colitis evaluated. Mice received P28GST-loaded or P28GST-free PLGA microparticles (s.c.) on Day 0, and optionally also on Days 14 and 28. Colitis was induced on Day 35, the animals were sacrificed on Day 37. Interestingly, the Wallace score (being a measure of the severity of the inflammation) was significantly lower in mice treated with P28GST microparticles compared to placebo after 1 or 3 injections. As immunogenicity markers, increased anti-P28GST IgG levels were detected after three P28GST PLGA microparticle injections, but not in the control groups. Thus, the proposed microparticles offer an interesting potential for the preventive treatment of experimental colitis, while the underlying mechanism of action is still to be investigated.

Structural and enzymatic insights into species-specific resistance to schistosome parasite drug therapy.

The antischistosomal prodrug oxamniquine is activated by a sulfotransferase (SULT) in the parasitic flatworm Schistosoma mansoni. Of the three main human schistosome species, only S. mansoni is sensitive to oxamniquine therapy despite the presence of SULT orthologs in Schistosoma hematobium and Schistosoma japonicum The reason for this species-specific drug action has remained a mystery for decades. Here we present the crystal structures of S. hematobium and S. japonicum SULTs, including S. hematobium SULT in complex with oxamniquine. We also examined the activity of the three enzymes in vitro; surprisingly, all three are active toward oxamniquine, yet we observed differences in catalytic efficiency that implicate kinetics as the determinant for species-specific toxicity. These results provide guidance for designing oxamniquine derivatives to treat infection caused by all species of schistosome to combat emerging resistance to current therapy.

Defining the Schistosoma haematobium kinome enables the prediction of essential kinases as anti-schistosome drug targets.

The blood fluke Schistosoma haematobium causes urogenital schistosomiasis, a neglected tropical disease (NTD) that affects more than 110 million people. Treating this disease by targeted or mass administration with a single chemical, praziquantel, carries the risk that drug resistance will develop in this pathogen. Therefore, there is an imperative to search for new drug targets in S. haematobium and other schistosomes. In this regard, protein kinases have potential, given their essential roles in biological processes and as targets for drugs already approved by the US Food and Drug Administration (FDA) for use in humans. In this context, we defined here the kinome of S. haematobium using a refined bioinformatic pipeline. We classified, curated and annotated predicted kinases, and assessed the developmental transcription profiles of kinase genes. Then, we prioritised a panel of kinases as potential drug targets and inferred chemicals that bind to them using an integrated bioinformatic pipeline. Most kinases of S. haematobium are very similar to those of its congener, S. mansoni, offering the prospect of designing chemicals that kill both species. Overall, this study provides a global insight into the kinome of S. haematobium and should assist the repurposing or discovery of drugs against schistosomiasis.

Comparative evaluation of Schistosoma mansoni, Schistosoma intercalatum, and Schistosoma haematobium alkaline phosphatase antigenicity by the alkaline phosphatase immunoassay (APIA).

To know if alkaline phosphatase (AP) from schistosomes other than Schistosoma mansoni can be used as diagnostic marker for schistosomiasis in alkaline phosphatase immunocapture assay (APIA), we comparatively tested n-butanol extracts of adult worm membranes from a Venezuelan (JL) strain of S. mansoni (Ven/AWBE/Sm); a Cameroonian (EDEN) strain of Schistosoma intercalatum (Cam/AWBE/Si) and a Yemeni strain of Schistosoma haematobium (Yem/AWBE/Sh). APIA was evaluated with sera of patients from Venezuela, Senegal, and Gabon infected with S. mansoni, from Gabon infected with S. intercalatum or S. haematobium, from Chine infected with Schistosoma japonicum and from Cambodian patients infected with Schistosoma mekongi. Results indicate that 92.5% (37/40) of Venezuela sera, 75% (15/20) of Senegal sera, 39.5% (17/43) of S. haematobium sera, and 19.2% (5/26) S. intercalatum sera were APIA-positive with the Ven/AWBE/Sm preparation. APIA with the Cam/AWBE/Si preparation showed that 53.8% of S. intercalatum-positive sera had anti-AP antibodies, and 51.2% S. haematobium-positive sera cross-immunocapturing the S. intercalatum AP. APIA performed with Yem/AWBE/Sh showed that 55.8% S. haematobium sera were positive. Only two out of nine S. japonicum sera were APIA-positive with the Ven/AWBE/Sm and Cam/AWBE/Si, and no reaction was observed with Cambodian S. mekongi-positive sera. AP activity was shown to be present in all the schistosome species/strains studied. The use of APIA as a tool to explore the APs antigenicity and the presence of Schistosoma sp. infections through the detection of anti-Schistosoma sp. AP antibodies in a host, allowed us to demonstrate the antigenicity of APs of S. mansoni, S. intercalatum, and S. haematobium.

Enzymatic activity and immunolocalization of Schistosoma mansoni and Schistosoma haematobium neutral sphingomyelinase.

We predicted, and provided evidence for, the existence of a schistosome tegument-associated Mg(2+)-dependent neutral sphingomyelinase (nSMase), which controls hydrolysis of surface membrane sphingomyelin molecules, thus allowing nutrients, but not host antibodies, to access proteins at the host-parasite interface. While a putative nSMase was identified in a recent Schistosoma mansoni genome sequencing and analysis study, our report is the first to measure nSMase enzymatic activity in Triton X-100-solubilized surface membrane (Sup 1) and whole worm soluble (SWAP) molecules of male and female S. mansoni and Schistosoma haematobium. Neutral, but no acidic, sphingomyelinase activity was readily detectable by the amplex red sphingomyelinase assay, and increased with incubation time and protein amount. Like nSMase family members, the schistosome nSMase activity was significantly (P<0.05 to <0.0001) enhanced by unsaturated fatty acids and phosphatidyl serine and significantly (P<0.01) decreased following exposure to the nSMase specific inhibitor GW4869. Peptides based on the published sequence of S. mansoni putative nSMase and used in a multiple antigen peptide form induced the generation of specific antibodies, which readily bound to the immunogen and to the cognate protein in Sup 1 and SWAP. Immunofluorescence studies suggested the parasite nSMase is located in the worm tegument and gut lining. Studies using RNA interference are in progress to define nSMase role in larval and adult worm surface membrane antigen exposure and unsaturated fatty acid-mediated attrition.

Conservation of protein kinase a catalytic subunit sequences in the schistosome pathogens of humans.

cAMP-dependent protein kinases (PKAs) are central mediators of cAMP signaling in eukaryotic cells. Previously we identified a cDNA which encodes for a PKA catalytic subunit (PKA-C) in Schistosoma mansoni (SmPKA-C) that is required for adult schistosome viability in vitro. As such, SmPKA-C could potentially represent a novel schistosome chemotherapeutic target. Here we sought to identify PKA-C subunit orthologues in the other medically important schistosome species, Schistosoma haematobium and Schistosoma japonicum, to determine the degree to which this potential target is conserved and could therefore be exploited for the treatment of all forms of schistosomiasis. We report the identification of PKA-C subunit orthologues in S. haematobium and S. japonicum (ShPKA-C and SjPKA-C, respectively) and show that PKA-C orthologues are highly conserved in the Schistosoma, with over 99% amino acid sequence identity shared among the three human pathogens we examined. Furthermore, we show that the recently published Schistosoma mansoni and S. japonicum genomes contain sequences encoding for several putative PKA substrates with homology to those found in Homo sapiens, Caenorhabditis elegans, and Saccharomyces cerevisiae.

Probing the mechanism of GSH activation in Schistosoma haematobium glutathione-S-transferase by site-directed mutagenesis and X-ray crystallography.

During turnover, the catalytic tyrosine residue (Tyr10) of the sigma class Schistosoma haematobium wild-type glutathione-S-transferase is expected to switch alternately in and out of the reduced glutathione-binding site (G-site). The Tyrout10 conformer forms a pi-cation interaction with the guanidinium group of Arg21. As in other similar glutathione-S-transferases, the catalytic Tyr has a low pKa of 7.2. In order to investigate the catalytic role of Tyr10, and the structural and functional roles of Arg21, we carried out structural studies on two Arg21 mutants (R21L and R21Q) and a Tyr10 mutant, Y10F. Our crystallographic data for the two Arg21 mutants indicate that only the Tyrout10 conformation is populated, thereby excluding a role of Arg21 in the stabilisation of the out conformation. However, Arg21 was confirmed to be catalytically important and essential for the low pKa of Tyr10. Upon comparison with structural data generated for reduced glutathione-bound and inhibitor-bound wild-type enzymes, it was observed that the orientations of Tyr10 and Arg35 are concerted and that, upon ligand binding, minor rearrangements occur within conserved residues in the active site loop. These rearrangements are coupled to quaternary rigid-body movements at the dimer interface and alterations in the localisation and structural order of the C-terminal domain.

Insights into the catalytic mechanism of glutathione S-transferase: the lesson from Schistosoma haematobium.

Glutathione S-transferases (GSTs) are involved in detoxification of xenobiotic compounds and in the biosynthesis of important metabolites. All GSTs activate glutathione (GSH) to GS(-); in many GSTs, this is accomplished by a Tyr at H-bonding distance from the sulfur of GSH. The high-resolution structure of GST from Schistosoma haematobium revealed that the catalytic Tyr occupies two alternative positions, one external, involving a pi-cation interaction with the conserved Arg21, and the other inside the GSH binding site. The interaction with Arg21 lowers the pK(a) of the catalytic Tyr10, as required for catalysis. Examination of several other GST structures revealed the presence of an external pocket that may accommodate the catalytic Tyr, and suggested that the change in conformation and acidic properties of the catalytic Tyr may be shared by other GSTs. Arginine and two other residues of the external pocket constitute a conserved structural motif, clearly identified by sequence comparison.

Schistosoma japonicum reveals distinct reactivity with antisera directed to proteases mediating host infection and invasion by cercariae of S. mansoni or S. haematobium.

Serine proteases released from the acetabular glands of cercariae, also known as cercarial elastases, are key enzymes in the penetration process of schistosomes through the skin of the final host. Antisera against these enzymes secreted from Schistosoma mansoni or S. haematobium reveal differences in the patterns of elastase expression among schistosome species and among different developmental stages of the larvae. Immunolocalization studies showed that antisera raised against the enzyme s28 protease react with S. mansoni, S. haematobium and also S. japonicum, in developing as well as mature cercariae and in both pre- and post-acetabular glands. Antisera against the enzyme SmCE detect the respective antigen solely in the pre-acetabular glands. Remarkably, the SmCE-1a isoform is detectable with DNA-vaccinated mouse sera in S. mansoni and S. haematobium only, but is apparently absent from the acetabular glands of S. japonicum. These differences in immunoreactivity of cercarial enzymes may be related to the distinct infection process of S. japonicum.

In vitro and in vivo effects of unsaturated fatty acids on Schistosoma mansoni and S. haematobium lung-stage larvae.

Incubation of Schistosoma mansoni lung-stage larvae in 90% corn oil for 6 hr was shown to elicit exposure of their, otherwise masked, apical membrane antigens to binding of anti-schistosome antibodies in the indirect membrane immunofluorescence test (IF). The possibility that unsaturated fatty acids (FA) are responsible for this effect was herein supported by IF data on ex vivo lung-stage larvae of S. mansoni and S. haematobium incubated for 1/2-2 hr with 80% corn oil, 50% olive oil, or 10-20 microM arachidonic acid. Treatment with unsaturated FA followed by filipin staining for cholesterol visualization indicated that unsaturated FA do not induce exposure of schistosomular surface membrane antigens via extraction of surface membrane cholesterol. Evidence using inhibitors and stimulators of neutral sphingomyelinase suggested that unsaturated FA perhaps activate worm tegument-bound neutral sphingomyelinase, leading to sphingomyelin hydrolysis and changes in surface membrane fluidity. Larval apical membrane antigens are, thus, allowed to diffuse freely in the plane of the membrane and bind specific antibodies in IE Excessive sphingomyelin hydrolysis might explain why high FA concentrations or long incubation periods eventually lead to larval death. The significant decrease (P < 0.01) in S. mansoni and increase (P < 0.02) in S. haematobium worm recovery in BALB/c mice given unsaturated FA-high and -poor diets, respectively, indicated these findings have in vivo relevance and led to the proposal that unsaturated FA likely plays a role in natural attrition of S. mansoni and S. haematobium lung-stage larvae.

Mapping and sequencing of acetylcholinesterase genes from the platyhelminth blood fluke Schistosoma.

Acetylcholinesterase (AChE) on the surface of the parasitic blood fluke Schistosoma is the likely target for schistosomicidal anticholinesterases. Determination of the molecular structure of this drug target is key for the development of improved anticholinesterase drugs and potentially a novel vaccine. We have recently cloned the cDNA encoding the AChE from the human parasite Schistosoma haematobium and succeeded in expressing functional recombinant protein. We now describe the cloning and molecular characterisation of homologues from two other schistosome species-Schistosoma mansoni and Schistosoma bovis, which are important parasites of man and cattle, respectively, but which differ in their sensitivity to the therapeutic anticholinesterase metrifonate. Comparison of the deduced amino acid sequences revealed that the AChE from all three species posses a high degree of identity, with conservation of all of the residues known to be important for substrate binding and catalytic activity. Also conserved is a unique C-terminal domain which is unusual in that it lacks the consensus for GPI modification, even though the native protein is considered to be GPI-anchored. We have also established the AChE gene structures for all three species and cloned the complete gene for S. haematobium AChE. The gene structure is relatively complex, comprising nine coding exons; the location of the splice sites is identical in all three species, but the size of the introns varies considerably. The two C-terminal splicing sites that are conserved in all species are also present in Schistosoma, but a third C-terminal conserved splicing site which is located 11-13 amino acids upstream of the histidine of the catalytic triad in all invertebrate AChE genes characterised to date is absent. We discuss our findings in the context of the molecular phylogeny of the AChE genes and the potential application to the control of schistosomiasis.

Crystal structure of the 28 kDa glutathione S-transferase from Schistosoma haematobium.

Schistomiasis is a debilitating parasitic disease which affects 200 million people, causing life-threatening complications in 10% of the patients. This paper reports the crystal structure of the Schistosoma haematobium 28 kDa glutathione S-transferase, a multifunctional enzyme involved in host-parasite interactions and presently considered as a promising vaccine candidate against schistosomiasis. The structures of the GSH-free enzyme, as well as the partially (approximately 40%) and almost fully (approximately 80%) GSH-saturated enzyme, exhibit a unique feature, absent in previous GST structures, concerning the crucial and invariant Tyr10 side chain which occupies two alternative positions. The canonical conformer, which allows an H-bond to be formed between the side chain hydroxyl group and the activated thiolate of GSH, is somewhat less than 50% occupied. The new conformer, with the phenoxyl ring on the opposite side of the mobile loop connecting strand 1 and helix 1, is stabilized by a polar interaction with the guanidinium group of the conserved Arg21 side chain. The presence of two conformers of Tyr10 may provide a clue about clarifying the multiple catalytic functions of Sh28GST and might prove to be relevant for the design of specific antischistosomal drugs. The K(d) for GSH binding was determined by equilibrium fluorescence titrations to be approximately 3 microM and by stopped-flow rapid mixing experiments to be approximately 9 microM. The relatively tight binding of GSH by Sh28GST explains the residually bound GSH in the crystal and supports a possible role of GSH as a tightly bound cofactor involved in the catalytic mechanism for prostaglandin D(2) synthase activity.

Isoenzyme analysis of Schistosoma haematobium, S. intercalatum and their hybrids and occurrences of natural hybridization in Cameroon.

Isoelectric focusing of glucose-6-phosphate dehydrogenase (G6PD) produced clearly identifiable profiles for S. haematobium and S. intercalatum and their hybrids. To provide a more detailed analysis of the interactions of S. haematobium and S. intercalatum in South West Cameroon over the last 12 years, G6PD analyses were carried out on individual schistosomes collected in Kumba in 1990, Loum in 1990, 1999 and 2000 and Barombi Mbo and Barombi Kotto in 1999. Studies were also carried out on the two parental species S. haematobium Barombi Mbo, S. intercalatum Edea and subsequent generations of hybrids resulting from laboratory crosses of the two parental species. The isoenzyme analysis demonstrated that the 1990 isolate from Kumba, was a recombinant of S. intercalatum x S. haematobium, and that 30% of individual schistosomes collected in 1990 in Loum were also recombinants. The remainder gave data indicative of S. haematobium. In 1999, 12.5% of individuals from Loum showed recombination and 10% in 2000. Results from the most recent parasitological survey in October 2000 showed the persistence of the recombinant population in addition to that of S. haematobium. There was also evidence of recombination having taken place in Barombi Kotto but not Barombi Mbo. This study demonstrates how the situation has changed over the last 12 years, and emphasizes the importance of assessing morphological, biological and molecular data together to gain a true picture of the rapidly evolving situation.

Evidence for the presence of active cytochrome P450 systems in Schistosoma mansoni and Schistosoma haematobium adult worms.

Extracts of the adult worms of both Schistosoma mansoni and Schistosoma haematobium can metabolise some typical P450 substrates but to differing degrees. S. mansoni worm extracts displayed a approximately 12-fold higher specific activity for an aminopyrine substrate than rat liver microsomes. At 4 mM substrate concentration the demethylation reaction with N-nitrosodimethylamine (NDMA) (5 nmol HCHO/mg protein/min) was only half that of rat liver microsomes, whereas in extracts of S. haematobium, no detectable activity was found towards NDMA. Using ethylmorphine as substrate the demethylation activity of S. mansoni extracts (1.82 nmol HCHO/mg protein/min) was 5.5-fold lower than that of rat liver microsomes. Benzphetamine demethylase activity was also readily detectable in S. mansoni worm extracts at 6.79 nmol HCHO/mg protein/min compared with 10.20 nmol HCHO/mg protein/min in the case of rat liver microsomes. When aniline was used as substrate, surprisingly, no activity was found in worm extracts of either S. mansoni or S. haematobium, whereas rat liver microsomes showed high activity towards this amine. The anti-P450 2E1 and 2B1/2 cross-reacted with both worm homogenates and gave a specific band corresponding to a protein of molecular weight of approximately 50.0 kDa. A study with anti-P450 IVA antibody revealed that while this protein was strongly expressed in S. haematobium worm extracts, no immunoreactivity was observed with extracts of S. mansoni. Immunoblotting analyses with anti-P450 IIIA and P450 1A1 did not detect immunoreactive protein in either S. mansoni or S. haematobium.

Molecular characterization of an acetylcholinesterase implicated in the regulation of glucose scavenging by the parasite Schistosoma.

Acetylcholinesterase (AChE) present on the surface of the trematode blood fluke Schistosoma has been implicated in the regulation of glucose scavenging from the host blood. Determination of the molecular structure and functional characteristics of this molecule is a crucial first step in understanding the novel function for AChE and in evaluating the potential of schistosome AChE as a target of new parasite control methods. We have determined the primary structure of acetylcholinesterase from Schistosoma haematobium. Immunolocalization studies confirmed that the enzyme was present on the parasite surface as well as in the muscle. The derived amino acid sequence possesses features common to acetylcholinesterases: the catalytic triad, six cysteines that form three intramolecular disulphide bonds, and aromatic residues lining the catalytic gorge. An unusual feature is that the fully processed native enzyme exists as a glycoinositol phospholipid (GPI)-anchored dimer, but the sequence of the C?terminus does not conform to the current consensus for GPI modification. The enzyme expressed in Xenopus oocytes showed conventional substrate specificity and sensitivity to established inhibitors of AChE, although it is relatively insensitive to the peripheral site inhibitor propidium iodide. Distinctions between host and parasite AChEs will allow the rational design of schistosome-specific drugs and vaccines.

Functional specific binding of testosterone to Schistosoma haematobium 28-kilodalton glutathione S-transferase.

During parasitic disease such as schistosomiasis, sex hormones have an important influence on the age- and gender-dependent level of infection. Since mammal glutathione S-transferase (GST) has the ability to bind hormones and particularly sexual steroids to influence their transport, metabolism, and physiological action, we have evaluated the capacity of testosterone to bind the 28-kDa GST of the Schistosoma haematobium parasite (Sh28GST). For the first time, we have demonstrated a specific binding of testosterone to parasite GST protein with high affinity (K(d) = 2.57 x 10(-7) M). In addition, we have assessed the effect of this binding on Sh28GST enzymatic activity, a mechanism closely associated with the reduction of Schistosoma fecundity. We showed that testosterone has the functional ability to inhibit the Sh28GST enzymatic activity in a dose-dependent manner, suggesting that this hormone could be directly involved in an antifecundity mechanism. This effect seemed to be related to the binding of testosterone to one peptide involved in the enzymatic site (i.e., amino acids 24 to 43). During human infection, binding of sexual hormones to Schistosoma Sh28GST could play a key role in parasite metabolism, especially the decrease of fecundity, and could be involved in the sex-dependent immune response to Sh28GST that we have previously observed in infected adults.

Features of the antibody response attributable to plasmid backbone adjuvanticity after DNA immunization.

DNA vaccination induces antigen-specific immune responses with characteristics distinct from other vaccination modes. In the present study, the contribution of the plasmid backbone adjuvant effect to the quality of the DNA-raised antibody response was investigated. For this purpose, three intradermal primings were compared in mice using: (1) the recombinant Schistosoma haematobium glutathione S-transferase antigen (rSh28GST): (2) rSh28GST supplemented with a non-coding plasmid; and (3) a Sh28GST-encoding plasmid. In contrast to immunization with the protein, DNA immunization elicited a very stable antibody (Ab) response over a prolonged period of time. This feature was attributed to the plasmid backbone, because co-administration of the non-coding plasmid with rSh28GST allowed the maintenance of the specific Ab response. A strong anamnestic Ab response was induced after intradermal boost with rSh28GST only in the mice primed with pMSh. This indicated that the selective ability of DNA vaccination to induce memory humoral response was independent of the plasmid backbone. In contrast the plasmid backbone was found to strongly participate in the preferential IgG2a Ab production observed. These results suggest that, following DNA immunization, the Th1-biased profile and the maintenance of the long-lived Ab response could be attributed to an adjuvant effect of the plasmid backbone during priming, whereas the strength of B-cell memory was independent of this effect.