Prevalence and Associated Risk Factors of Urinary Schistosomiasis among Primary School Pupils in the Jidawa and Zobiya Communities of Jigawa State, Nigeria.

Background: Urogenital schistosomiasis (UgS) is a parasitic disease caused by Schistosoma haematobium and can lead to chronic ill-health. Nigeria is endemic for schistosomiasis, but epidemiology of UgS has not been studied in most states. This study was conceived with the aim to contribute towards an accurate national picture of UgS in Nigeria. The prevalence of UgS and the associated risk factors were for the first time investigated among primary school pupils in Jidawa and Zobiya communities of the Dutse Local Government Area (LGAs) of Jigawa State, Nigeria. Method: Focus group discussions with teachers and parents were conducted. After obtaining written consent from parents, questionnaires were administered to pupils to obtain socio-demographic data and information on water contact activities. Urine samples (279) were collected and processed by the urine filtration technique to evaluate haematuria and the presence of S. haematobium eggs. Results: Prevalences of 65.7% (90/137) and 69.0% (98/142) were recorded in the Jidawa and Zobiya communities, respectively. In both communities, there was a significant association between gender and UgS: 63.3% of the infected pupils were males as compared to 36.7% females (χ2 = 5.42, p = 0.020). Grade 5 students had a significantly higher prevalence (χ2 = 17.919, p = 0.001) (80.0%) compared to those in grades 2, 3, 4, and 6 (63.8%, 66.7%, 61.5%, and 64.6%, respectively). Water contact activities showed that pupils involved in fishing, irrigation, and swimming were at greater risk of becoming infected in Jidawa and Zobiya, with odds ratios (risk factors) of 5.4 (0.994-28.862) and 4.1 (1.709-9.862), respectively (p = 0.05). Conclusion: Both the Jidawa and Zobiya communities of the Dutse LGAs of Jigawa State are hyperendemic for UgS. In collaboration with the State Ministry of Health, mass administration of praziquantel was carried out in the Jidawa and Zobiya communities after this study.

Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.

Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.

Aryl ureas represent a new class of anti-trypanosomal agents.

BACKGROUND: The trypanosomal diseases including Chagas' disease, African sleeping sickness and Nagana have a substantial impact on human and animal health worldwide. Classes of effective therapeutics are needed owing to the emergence of drug resistance as well as the toxicity of existing agents. The cysteine proteases of two trypanosomes, Trypanosoma cruzi (cruzain) and Trypanosoma brucei (rhodesain), have been targeted for a structure-based drug design program as mechanistic inhibitors that target these enzymes are effective in cell-based and animal models of trypanosomal infection. RESULTS: We have used computational methods to identify new lead scaffolds for non-covalent inhibitors of cruzain and rhodesain, have demonstrated the efficacy of these compounds in cell-based and animal assays, and have synthesized analogs to explore structure activity relationships. Nine compounds with varied scaffolds identified by DOCK4.0.1 were found to be active at concentrations below 10 microM against cruzain and rhodesain in enzymatic studies. All hits were calculated to have substantial hydrophobic interactions with cruzain. Two of the scaffolds, the urea scaffold and the aroyl thiourea scaffold, exhibited activity against T. cruzi in vivo and both enzymes in vitro. They also have predicted pharmacokinetic properties that meet Lipinski's 'rule of 5'. These scaffolds are synthetically tractable and lend themselves to combinatorial chemistry efforts. One of the compounds, 5'(1-methyl-3-trifluoromethylpyrazol-5-yl)-thiophene 3'-trifluoromethylphenyl urea (D16) showed a 3.1 microM IC(50) against cruzain and a 3 microM IC(50) against rhodesain. Infected cells treated with D16 survived 22 days in culture compared with 6 days for their untreated counterparts. The mechanism of the inhibitors of these two scaffolds is confirmed to be competitive and reversible. CONCLUSIONS: The urea scaffold and the thiourea scaffold are promising leads for the development of new effective chemotherapy for trypanosomal diseases. Libraries of compounds of both scaffolds need to be synthesized and screened against a series of homologous parasitic cysteine proteases to optimize the potency of the initial leads.

Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris.

Asparaginyl endopeptidases, or legumains, are a recently identified family of cysteine-class endopeptidases. A single gene encoding a Schistosoma mansoni asparaginyl endopeptidase (a.k.a. Sm32 or schistosome legumain) has been reported, but by sequence homology it would be expected to yield an inactive product as the active site C197 had been replaced by N. We now describe a new S. mansoni gene in which C197 is present. Both gene products were expressed in Pichia pastoris. Autocatalytic processing to fully active C197 Sm32 occurred at acid pH. In contrast, N197 Sm32 was not processed and this is consistent with the hypothesis that C197 is essential for catalysis. This was confirmed by mutation of N197 to C and re-expression in Pichia. The availability of recombinant active Sm32 allows detailed analysis of its catalytic mechanism and its function(s) in the biology of this important human parasite.

Cysteine proteinases of trypanosome parasites: novel targets for chemotherapy.

The protozoan parasites, Trypanosoma brucei and T. cruzi, that cause sleeping sickness in sub-Saharan Africa and Chagas' Disease in Latin America, respectively, exert significant morbidity and mortality in man. Combinations of toxicity and differential efficacy of current drugs provide an urgent need to develop novel, cheap and effective chemotherapies. Research over the last decade with cultured trypanosomes and mice experimentally infected with these parasites has demonstrated that trypanosome cysteine proteinases are valid targets for the rational design of new drugs. In particular, potent peptidyl and peptidomimetic inhibitors of brucipain (a.k.a. trypanopain-Tb) and cruzain (a.k.a. cruzipain), the respective cysteine proteinases of T. brucei and T. cruzi, have proved trypanocidal. Efforts are ongoing to develop more specific non-toxic inhibitors of various chemistries with improved biological half-lives and biovailability characteristics. Here, the biochemical and biological properties together with the history, current status and perceived directions on the development of specific inhibitors of trypanosome cysteine proteinases will be reviewed.

Schistosoma japonicum is less sensitive to cyclosporin A in vivo than Schistosoma mansoni.

We compared the toxic effects of cyclosporin A (CsA) on postmigratory immature Schistosoma mansoni and Schistosoma japonicum in mice. For each species, CsA was administered either relatively early or late during development. Exposure of 20-day-old S. mansoni to 1 subcutaneous dose of CsA (50 mg/kg) reduced the worm burden by 45% and induced herniae and/or boli of the gut in 32% of perfusable worms. These results agree with previous reports. In addition, CsA induced a marked liver shift (37% of total worm number). For S. japonicum, CsA was administered at 11 days postinfection (PI) because this species migrates more quickly. Killing of worms and damage to the gut were not observed, and only a slight liver shift occurred. Similarly, these effects were not recorded when CsA was administered at the later times of 34 days PI for S. mansoni and 17 days PI for S. japonicum. For both species, CsA stunted worms, affecting both sexes early PI but only females late PI. In conclusion, immature worms of S. japonicum are less sensitive than S. mansoni to CsA. Also, S. mansoni displays marked age-dependent differences in its sensitivity to CsA.

Cysteine protease inhibitors as chemotherapy for parasitic infections.

Analysis of the evolution, localization and biologic function of papain family cysteine proteases in metazoan and protozoan parasites has provided important and often surprising insights into the biochemistry and cellular function of this diverse enzyme family. Furthermore, the relative lack of redundancy of cysteine proteases in parasites compared to their mammalian hosts makes them attractive targets for the development of new antiparasitic chemotherapy. The treatment of experimental models of parasitic diseases with cysteine protease inhibitors has provided an important 'proof of concept' for the use of cysteine protease inhibitors in vivo. Evidence has now accumulated that cysteine protease inhibitors can selectively arrest replication of a microbial pathogen without untoward toxicity to the host. Furthermore, this can be achieved with reasonable dosing schedules and oral administration of the drug. Initial studies have confirmed the efficacy of cysteine protease inhibitors in treatment of Trypanosoma cruzi, Plasmodium falciparum and Leishmania major. Work on Trypanosoma brucei, the agent of African trypanosomiasis, is preliminary but also promising. Target validation studies have shown that biotinylated or radiolabeled irreversible inhibitors specifically bind to the cysteine protease targets thought to represent the major activity within the parasite. In the case of T. cruzi, the effect of inhibitors appears to be predominantly in blocking protease processing. Transfection studies using variant constructs have supported this model. Finally, the generation of null mutants for the multiple protease genes in Leishmania mexicana has provided the first genetic support for the key role of this enzyme family in parasite virulence. Safety studies in rodents and analysis of uptake of inhibitors by parasites and host cells suggest that the selectivity of inhibitors for the parasite targets may reside in the lack of redundancy of parasite proteases, the higher concentration of host proteases in intracellular compartments, and differential uptake of inhibitors by parasites. Attempts to elicit resistance to cysteine protease inhibitors in parasite cultures suggest that mechanisms of induced resistance are independent of resistance to the traditional antiparasitic agents. This suggests that cysteine protease inhibitors may provide an alternative to traditional therapy in drug-resistant organisms.

Trypanosoma brucei: killing of bloodstream forms in vitro and in vivo by the cysteine proteinase inhibitor Z-phe-ala-CHN2.

Cysteine proteinases were tested for their suitability as targets for chemotherapy of sleeping sickness using the peptidyl inhibitor Z-Phe-Ala-diazomethyl ketone (Z-Phe-Ala-CHN2). In vitro, the inhibitory concentration of Z-Phe-Ala-CHN;2 required to reduce the growth rate by 50% was 400 times lower for culture-adapted bloodstream forms of Trypanosoma brucei than for a mouse myeloma cell line. At an inhibitor concentration of 10;M the parasites were lysed within 48 h of incubation. Parasitemia of mice infected with T. brucei decreased to undetectable levels for 3 days following treatment with 250 mg/kg Z-Phe-Ala-CHN2 on days 3 to 6 after infection. Although parasitemia returned thereafter to control levels, infected mice treated with the inhibitor survived approximately twice as long as those treated with placebo. Z-Phe-Ala-CHN2 inhibited proteinolysis in lysosomes in vitro and almost completely blocked cysteine proteinase activity in vivo. The results demonstrate the importance of cysteine proteinase activity for survival of T. brucei and suggest that such activity is an appropriate target for antitrypanosomal chemotherapy.

Schistosoma haematobium, S. intercalatum, S. japonicum, S. mansoni, and S. rodhaini in mice: relationship between patterns of lung migration by schistosomula and perfusion recovery of adult worms.

The development of five schistosome species was compared in mice by the recovery of schistosomula from chopped lung tissue and of adult worms by portal perfusion. Three developmental patterns appeared. (1) Schistosoma japonicum was unique in showing an early establishment of schistosomula in and a rapid departure from the lungs together with the highest worm recovery; (2) S. haematobium contrasted by establishing later and persisting in the lungs for at least 2 weeks while yielding the lowest adult worm recovery; and (3) S. intercalatum, S. mansoni, and S. rodhaini had an intermediate pattern--they resided in the lungs for several days, then disappeared and produced intermediate numbers of adults. Lung petechiae, known to accompany the migration of S. japonicum, were never detected after infection with the other species. We speculate that the three migration patterns of schistosomes are related to the size of the relative spectra of naturally infected definitive hosts.

Affinity isolation and characterization of the cathepsin B-like proteinase Sj31 from Schistosoma japonicum.

The major cathepsin B-like proteinase of adult Schistosoma japonicum has been isolated for the first time. Affinity chromatography with the mammalian cathepsin B inhibitor glycyl-phenylalanyl-glycine-semicarbazone purified a protein that was identified by N-terminal sequencing as Sj31. Sensitivity of Sj31 to PNGase F demonstrated the presence of asparagine-linked N-glycan. Marked resistance to the action of Endo-beta-glycosidase H indicated that most of the N-glycan chains are of the complex type. Binding of horseradish peroxidase-conjugated lectins demonstrated the presence of N-mannose, N-acetylglucosamine, and N-acetyllactosamine type 2 in the N-glycan. Fucose was not detected, and the presence of sialic acid remained questionable. Sj31 degraded the fluorogenic substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec with an optimum between pH 5.0 and 6.0. The specific activity was 18-21-fold higher with the Phe-Arg substrate compared with the Arg-Arg substrate, whereas this value was 4-6-fold for bovine spleen cathepsin B, thus suggesting differences in the S2 subsite between parasite and host proteinases. Quantitative purification of Sj31 led to the conclusion that cathepsin B-like activity predominates over cathepsin L-like activity in S. japonicum. Because Sj31 degraded hemoglobin in vitro and was localized in the parasite gut, the proteinase may degrade ingested proteins in vivo.

Cathepsin B-like activity predominates over cathepsin L-like activity in adult Schistosoma mansoni and S. japonicum.

Both cathepsin B-like and cathepsin L-like endopeptidase activities have been described in schistosomes, but their relative contribution to proteinolysis remains controversial. In an attempt to clarify which type of activity predominates, the selective mammalian cathepsin B inhibitor CA-074 was tested under standardized assay conditions with different preparations from Schistosoma mansoni and S. japonicum. CA-074 (0.94 microM) inhibited at least 92% and 80% of proteinolytic activity, respectively, for these species: completely inhibited bovine-spleen cathepsin B activity; but showed only marginal inhibition (4%) of rat-liver cathepsin L activity. We discuss the results with respect to previous studies and conclude that schistosome cathepsin B-like, not L-like, activity predominates.

Schistosoma japonicum, S. mansoni, S. haematobium, S. intercalatum, and S. rodhaini: cysteine-class cathepsin activities in the vomitus of adult worms.

Vomitus from adults of five Schistosoma species was screened for biochemical homologues of the mammalian cysteine proteinases cathepsins B, H, and L. Bovine cathepsin B and rat cathepsin L served as references. Using the substrate Arg-NMec, a schistosome cathepsin H-like activity was never detected. All species degraded the cathepsin B substrate Z-Arg-Arg-NMec, but distinct species differences were observed with respect to pH optima and buffer preferences. The cathepsin B and L substrate Z-Phe-Arg-NMec was similarly degraded by all species, and activity was abolished by the cysteine proteinase inhibitor E-64. Preferences by vomitus proteinase activities for Z-Phe-Arg-NMec over Z-Arg-Arg-NMec were similar to or higher than those found for bovine cathepsin B but well below those observed for rat cathepsin L; also, the preferential cathepsin L inhibitor Z-Phe-PheCHN2 only partially inhibited proteinolytic activity. The results suggest the possible presence in vomitus of a minor cathepsin L-like activity and demonstrate a major cathepsin B-like activity that is biochemically variable between schistosome species.

Characterisation of proteolytic activity of excretory-secretory products from adult Strongylus vulgaris.

An excretory-secretory (ES) preparation derived from adult Strongylus vulgaris in vitro was assessed for proteolytic activity using azocasein and synthetic, fluorogenic, peptide substrates. Fractionation was by molecular sieve fast protein liquid chromatography (molecular sieve FPLC) and resolution by gelatin-substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (gelatin-substrate SDS-PAGE). The cysteine proteinase activator, dithiothreitol (DTT), enhanced azocaseinolysis and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NMec) by the ES preparation and was a requirement for the detection of carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NMec) hydrolysis. Assays of FPLC-eluted fractions, with DTT, detected a broad peak of azocaseinolytic activity (22-24 kDa) and two peaks (24 and 18 kDa) of hydrolysis using the synthetic substrates. Hydrolysis by these peaks of Z-Phe-Arg-NMec was 50-fold greater than that of Z-Arg-Arg-NMec suggesting that their specificities are more like papain or cathepsin L rather than cathepsin B. In gelatin-substrate SDS-PAGE, DTT was required to detect proteolysis by the ES preparation which was optimal at pH 6.0 and resolved into eight bands (87-29 kDa). Cysteine proteinase inhibitors were the most effective in all assays. Collectively, these data indicate that cysteine-class proteolytic activity predominates in the ES preparation of adult S. vulgaris.