Transgenesis in parasitic helminths: a brief history and prospects for the future.

Helminth infections impact the health of hundreds of millions of persons globally and also cause important economic losses in livestock farming. Methodological limitations as well as the low attention given to the study of helminths have impacted biological research and, thus, the procurement of accurate diagnosis and effective treatments. Understanding the biology of helminths using genomic and proteomic approaches could contribute to advances in understanding host-helminth interactions and lead to new vaccines, drugs and diagnostics. Despite the significant advances in genomics in the last decade, the lack of methodological adaptation of current transgenesis techniques has hampered the progression of post-genomic research in helminthology. However, the application of new techniques, such as CRISPR, to the study of trematodes and nematodes has opened new avenues for genome editing-powered functional genomics for these pathogens. This review summarises the historical advances in functional genomics in parasitic helminths and highlights pending limitations that will need to be overcome to deploy transgenesis tools.

Helminth Microbiomes - A Hidden Treasure Trove?

There is increasing attention on the complex interactions occurring between gastrointestinal parasitic helminths and the microbial flora (microbiota) inhabiting the host gut. However, little is known about the occurrence, structure, and function of microbial populations residing within parasite organs and tissues. In this article, we argue that an in-depth understanding of the interplay between parasites and their microbiomes may significantly enhance current knowledge of parasite biology and physiology, and may lead to the discovery of entirely novel, anthelmintic-independent interventions against parasites and parasitic diseases.

Immunization and challenge shown by hamsters infected with Opisthorchis viverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke.

Here we report findings to optimize and standardize conditions to attenuate metacercariae of Opisthorchis viverrini by ionizing radiation to elicit protective immune responses to challenge infection. Metacercariae were gamma-irradiated and the ability of irradiated metacercariae to prevent patent infection of challenge metacercariae in hamsters was determined, as well as their ability to induce a host antibody response. Metacercariae irradiated in a dose-dependent manner, with 3, 5, 10, 12, 20, 25 and 50 Gray, were used to infect Syrian golden hamsters by stomach gavage to ascertain the effect of irradiation on ability of the worms to establish infection. In addition, other hamsters were infected with metacercariae irradiated with 20-50 Gray, followed by challenge with intact/wild-type (non-irradiated) metacercariae to determine the protective effect as established by the numbers of adult flukes, eggs of O. viverrini in hamster faeces and anti-O. viverrini antibody titres. Significantly fewer worms were recovered from hamsters immunized with metacercariae irradiated at 20, 25 and 50 Gray than from control hamsters infected with intact metacercariae or 0 Gray, and the worms showed damaged reproductive organs. Faecal egg numbers were decreased significantly in hamsters immunized with 25 and 50 Gray metacercariae of O. viverrini. Moreover, hamsters administered metacercariae that were protected elicited a robust, specific anti-fluke immunoglobulin G response compared to control hamsters, suggesting a role for antibody in protection elicited by radiation-attenuated metacercariae.

Susceptibility of Snails to Infection with Schistosomes is influenced by Temperature and Expression of Heat Shock Proteins.

The freshwater snail, Biomphalaria glabrata is the obligate intermediate host for the transmission of the parasitic trematode, Schistosoma mansoni the causative agent of the chronic debilitating neglected tropical disease, schistosomiasis. We showed previously that in juvenile snails, early and significant induction of stress manifested by the expression of stress proteins, Hsp 70, Hsp 90 and reverse transcriptase (RT) of the non- LTR retrotransposon, nimbus, is a characteristic feature of juvenile susceptible NMRI but not resistant BS-90 snails. These latter, however, could be rendered susceptible after mild heat shock at 32°C, revealing that resistance in the BS-90 resistant snail to schistosomes is a temperature dependent trait. Here we tested the hypothesis that maintenance of BS-90 resistant snails at the permissive temperature for several generations affects the resistance phenotype displayed at the non-permissive temperature of 25°C. The progeny of BS-90 snails bred and maintained through several generations (F1 to F4) at 32°C were susceptible to the schistosome infection when returned to room temperature, shedding cercariae at four weeks post-infection. Moreover, the study of expression levels of the heat shock protein (Hsp) 70 protein by ELISA and western blot analysis, showed that this protein is also differentially expressed between susceptible and resistant snails, with susceptible snails expressing more protein than their resistant counterparts after early exposure to wild-type but not to radiation-attenuated miracidia. These data suggested that in the face of global warming, the ability to sustain a reduction in schistosomiasis by using refractory snails as a strategy to block transmission of the disease might prove challenging since non-lethal elevation in temperature, affects snail susceptibility to S. mansoni.

Comparison of findings using ultrasonography and cystoscopy in urogenital schistosomiasis in a public health centre in rural Angola.

BACKGROUND: Schistosomiasis is a chronic disease caused by infection with parasitic worms of the genus Schistosoma. In sub-Saharan Africa, infections with S. haematobium are most common. Cystoscopic examination (CE) has been accepted as the goldstandard test for detecting the late manifestations of schistosomiasis, including urothelial cancer of the bladder. However, this procedure is invasive and 10-40% of tumours may remain undetected. A non-invasive examination and a new generation of biomarkers are needed for better monitoring of the disease. OBJECTIVE: To assess the usefulness of ultrasound (US) scans for monitoring of structural urinary tract disease by local public health services in areas of Angola in which urogenital schistosomiasis is endemic. METHODS: A cohort of 80 S. haematobium-infected patients was selected in order to compare changes in the bladder wall detected by US with those observed on CE. RESULTS: There was a notable correlation between the findings observed on CE and US. Patients with lesions of the bladder mucosa such as neoplasms, ulcers or granulomas detected by CE also had changes in bladder wall thickness on US. The results support increased use of portable US machines for non-invasive examination of the bladder by local general practitioners. CONCLUSION: US examination should be an integral part of the investigation of haematuria and used in all S. haematobium control programmes. General practitioners may find it useful for more accurate diagnosis of haematuria and to identify bladder wall alterations in both adults and children in schistosomiasis-endemic regions.

Transgenesis of schistosomes: approaches employing mobile genetic elements.

Draft genome sequences for Schistosoma mansoni and Schistosoma japonicum are now available. However, the identity and importance of most schistosome genes have yet to be determined. Recently, progress has been made towards the genetic manipulation and transgenesis of schistosomes. Both loss-of-function and gain-of-function approaches appear to be feasible in schistosomes based on findings described in the past 5 years. This review focuses on reports of schistosome transgenesis, specifically those dealing with the transformation of schistosomes with exogenous mobile genetic elements and/or their endogenous relatives for the genetic manipulation of schistosomes. Transgenesis mediated by mobile genetic elements offers a potentially tractable route to introduce foreign genes to schistosomes, a means to determine the importance of schistosome genes, including those that could be targeted in novel interventions and the potential to undertake large-scale forward genetics by insertional mutagenesis.

Epidemiology of microsporidiosis: sources and modes of transmission.

Microsporidia are single-celled, obligate intracellular parasites that were recently reclassified from protozoa to fungi. Microsporidia are considered a cause of emerging and opportunistic infections in humans, and species infecting humans also infect a wide range of animals, raising the concern for zoonotic transmission. Persistent or self-limiting diarrhea are the most common symptoms associated with microsporidiosis in immune-deficient or immune-competent individuals, respectively. Microsporidian spores appear to be relatively resistant under environmental conditions, and species of microsporidia infecting humans and animals have been identified in water sources, raising concern about water-borne transmission. Sensitive and specific immunomagnetic bead separation and PCR-based methods are being developed and applied for detecting microsporidia in infected hosts and water sources for generating more reliable prevalence data. The most effective drugs for treating microsporidiosis in humans currently include albendazole, which is effective against the Encephalitozoon species but not against Enterocytozoon bieneusi, and fumagillin, which has broader anti-microsporidia activity but is toxic in mammals, suggesting a need to identify better drugs. Strategies to capture and disinfect microsporidia in water are being developed and include filtration, coagulation, chlorination, gamma-irradiation, and ozonation.

Hookworm cathepsin D aspartic proteases: contributing roles in the host-specific degradation of serum proteins and skin macromolecules.

Cathepsin D aspartic proteases of hookworms were recently implicated in the host-specific digestion of haemoglobin by adult parasites. Ac-APR-1 from the dog hookworm, Ancylostoma caninum and Na-APR-1 from the human hookworm, Necator americanus, were shown to be expressed in the infective larval stage (L3) as well as adult worms. We now show that both proteases degraded skin macromolecules and serum proteins, some of which were cleaved more readily from permissive definitive hosts as opposed to non-permissive hosts. Na-APR-1 degraded human collagens more efficiently than did Ac-APR-1, and Ac-APR-1 degraded canine serum albumin more efficiently than did Na-APR-1. On the other hand, both enzymes degraded human serum proteins (albumin and fibrinogen) with approximately equal efficiency under the conditions of our assays in vitro. Molecular models of these 2 orthologous, aspartic proteases showed that, despite having active site clefts with identical primary sequences, residues in the S3 pocket adopted different conformations, likely accounting for different substrate preferences reported previously. Antisera raised to both proteases partially inhibited (16-26%) migration of hookworm L3 through hamster skin in vitro, further implying a connective tissue invasive role for these enzymes in addition to digestion of serum and erythrocyte proteins for nutrition.

Hemoglobin-degrading, aspartic proteases of blood-feeding parasites: substrate specificity revealed by homology models.

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4' were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH(2)]Leu-Tyr-Tyr-Ser- NH(2) (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4' residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.

Receptor for Fc on the surfaces of schistosomes.

Schistosoma mansoni masks its surface with adsorbed host proteins including erythrocyte antigens, immunoglobulins, major histocompatibility complex class I, and beta(2)-microglobulin (beta(2)m), presumably as a means of avoiding host immune responses. How this is accomplished has not been explained. To identify surface receptors for host proteins, we biotinylated the tegument of live S. mansoni adults and mechanically transformed schistosomula and then removed the parasite surface with detergent. Incubation of biotinylated schistosome surface extracts with human immunoglobulin G (IgG) Fc-Sepharose resulted in purification of a 97-kDa protein that was subsequently identified as paramyosin (Pmy), using antiserum specific for recombinant Pmy. Fc also bound recombinant S. mansoni Pmy and native S. japonicum Pmy. Antiserum to Pmy decreased the binding of Pmy to Fc-Sepharose, and no proteins bound after removal of Pmy from extracts. Fluoresceinated human Fc bound to the surface, vestigial penetration glands, and nascent oral cavity of mechanically transformed schistosomula, and rabbit anti-Pmy Fab fragments ablated the binding of Fc to the schistosome surface. Pmy coprecipitated with host IgG from parasite surface extracts, indicating that complexes formed on the parasite surface as well as in vitro. Binding of Pmy to Fc was not inhibited by soluble protein A, suggesting that Pmy does not bind to the region between the CH2 and CH3 domains used by many other Fc-binding proteins. beta(2)m did not bind to the schistosome Fc receptor (Pmy), a finding that contradicts reports from earlier workers but did bind to a heteromultimer of labeled schistosomula surface proteins. This is the first report of the molecular identity of a schistosome Fc receptor; moreover it demonstrates an additional aspect of the unusual and multifunctional properties of Pmy from schistosomes and other parasitic flatworms.

Gut-associated immunolocalization of the Schistosoma mansoni cysteine proteases, SmCL1 and SmCL2.

Transcripts encoding discrete, cathepsin L-like cysteine proteases, known as SmCL1 and SmCL2, have been reported from the adult stages of the human blood fluke Schistosoma mansoni. However, the physiological roles of these 2 enzymes and their natural substrates remain uncertain and controversial. To determine their localization in adult S. mansoni by immunocytochemical procedures, and thereby to gain insight into their likely functions, polymerase chain reaction-based cDNAs encoding mature SmCL1 and SmCL2 were ligated into Escherichia coli. The bacterially expressed recombinant proteins (bSmSL1, bSmCL2) were used to generate monospecific rabbit antisera. For light microscopy, paraffin-embedded sections were visualized with the fluorophore Cy3. For transmission electron microscopy (TEM), LR White-embedded tissue was visualized with 15 nm gold. Under light microscopy, fluorescence was visible on the luminal surface of the gastrodermis in both sexes for both proteins. For bSmCL1 and bSmCL2, TEM revealed gold particles primarily associated with amorphous deposits within superficial digestive vacuoles on the gastrodermal surface of males and females. Some bSmCL1 reaction product was observed in vesicles within the gastrodermis, and very sparse gastrodermal activity was observed with bSmCL2. By contrast, neither enzyme was immunolocalized in the reproductive organs, vitelline glands, nor gynecorphoric canal. The gut-associated immunolocalization of SmCL1 and SmCL2 indicates that both these endopeptidases participate in hemoglobin proteolysis.

Schistosoma japonicum cathepsin D aspartic protease cleaves human IgG and other serum components.

Recombinant cathepsin D aspartic protease of Schistosoma japonicum cleaved human IgG in vitro in a time and dose-dependent manner. Optimal cleavage was seen at pH 3.6-4.5; modest cleavage remained at pH 5.0, and no cleavage was detected above pH 5.0. Amino terminal sequencing of the major cleavage fragments of human IgG identified a Fab fragment from the VH1 domain, and 2 cleavage sites in the CH2 domain below the hinge region. The P1 and P1' residues at the 2 CH2 cleavage sites were Phe254-Leu255 and Leu325-Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the immunoglobulin light chain was detected. In addition, the recombinant schistosome protease indiscriminately degraded the human serum proteins complement C3 and serum albumin into numerous small fragments. These results demonstrate specific cleavage of human IgG by the recombinant schistosome aspartic protease, and highlight the broad range digestive specificity of the enzyme which may play a role in the degradation of host serum proteins ingested as part of the schistosome bloodmeal.

Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum.

Mice were vaccinated with recombinant Schistosoma japonicum cathepsin D aspartic protease, expressed in both insect cells and bacteria, in order to evaluate the vaccine efficacy of the schistosome protease. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, we recorded increased egg output per female worm in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgG1, IgG2a and IgG2b isotypes), but there was no correlation between antibody levels and protective efficacy. Immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by the recombinant protease, and passive transfer of serum or antibodies from vaccinated animals, before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals. These results suggest that antibodies may not play a key role in the protective effect elicited, and that protection may be due to a combination of humoral and cell-mediated responses

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum.

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have named this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box. Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes.

Proteolysis of human hemoglobin by schistosome cathepsin D.

Schistosomes feed on human blood. They employ proteases to degrade hemoglobin from ingested erythrocytes, using the residues released for amino acid metabolism. However, the identity and the role of the participating protease(s) are unclear and controversial. Confocal microscopy localized schistosomal cathepsin D to the parasite gastrodermis, and revealed elevated protease expression in females. At sub-cellular level, cathepsin D was localized to superficial digestive vacuoles of the gut and to cisternae of the gastrodermal rough endoplasmic reticulum. Schistosome cathepsin D, expressed in insect cells, autoactivated at pH 3.6 to a approximately 40 kDa form that cleaved the substrates o-aminobenzoyl-Ile-Glu-Phe-nitroPhe-Arg-leu-NH(2) and hemoglobin. The NH(2)-terminal residues of mature cathepsin D of Schistosoma japonicum and Schistosoma mansoni were Asn1 and Gly1, respectively, revealing that the proregion peptide was comprised of 35 residues. The proteases cleaved hemoglobin at pH 2.5--4.6, releasing numerous fragments. S. Japonicum cathepsin D cleaved at 13 sites, S. mansoni cathepsin D at 15 sites. Early cleavage sites were alpha Phe33-Leu34 and beta Phe41-Phe42, while others included alpha Leu109-Ala-110 and beta Leu14-Trp15, demonstrating a preference for bulky hydrophobic residues at P1 and P1'. Most of the schistosomal cathepsin D cleavage sites were discrete from those of human cathepsin D. The gastrodermal location, elevated expression in females, acidic pH optima, similar substrate preferences in two species, and the discrete substrate preferences compared with human cathepsin D together provide compelling support for the hypothesis that schistosomal cathepsin D plays an integral role in hemoglobin proteolysis, and might be selectively targeted by drugs based on protease inhibition.

Purification of a diagnostic, secreted cysteine protease-like protein from the hookworm Ancylostoma caninum.

The enteric infection of humans with the canine hookworm Ancylostoma caninum varies in its clinical presentation, ranging from asymptomatic to eosinophilic gastroenteritis requiring surgical intervention. Infections are not patent, but can be diagnosed immunologically by detecting antibodies to an immunodominant secreted hookworm protein termed Ac68. To characterise Ac68, we purified the native protein from A. caninum excretory/secretory products using size exclusion followed by anion exchange chromatography. The epitopes in the purified protein recognised by human infection sera were shown to be proteins and not carbohydrates. The N-terminal amino acid sequence of the purified Ac68 was determined and six of the 11 residues obtained were shared with a previously characterised cysteine protease of A. caninum, AcCP1.

Sjalpha elements, short interspersed element-like retroposons bearing a hammerhead ribozyme motif from the genome of the oriental blood fluke Schistosoma japonicum.

Smalpha is a short interspersed element (SINE)-like retroposon that occurs in high copy number of the genome of the human blood fluke Schistosoma mansoni. The sequence of the consensus Smalpha element includes the hallmark features of SINE-like elements including a promoter region for RNA polymerase III, an AT-rich stretch at its 3'-terminus, a short length of 500 bp or less, and short direct repeat sequences flanking the insertion site. Interestingly, the sequence of Smalpha also encodes an active ribozyme bearing a hammerhead domain. Contrary to the recent findings of Ferbeyre et al. (Mol. Cell. Biol. 18 (1998) 3880-8) that indicated that Smalpha-like elements were absent from the genome of the Oriental blood fluke Schistosoma japonicum, we report here that the genome of S. japonicum does contain a family of Smalpha-like retroposons, elements that we have named the Sjalpha family. Like Smalpha, Sjalpha elements are SINE-like in structure and sequence, are present at high copy number interspersed throughout the S. japonicum genome, and contain an ostensibly functional, hammerhead ribozyme motif. The presence of these elements in all species of Schistosoma so far examined suggests that the hammerhead domain was acquired by vertical transmission from a common schistosome ancestor.

Molecular modeling and substrate specificity of discrete cruzipain-like and cathepsin L-like cysteine proteinases of the human blood fluke Schistosoma mansoni.

Adult Schistosoma mansoni blood flukes express two discrete cysteine proteinases, SmCL1 and SmCL2, both of which are related to the cathepsin L-like enzymes of the C1 family of peptidases. Our previous phylogenetic analysis indicated that SmCL1 is more closely related to cruzipain from the parasitic protozoa Trypanosoma cruzi than to human cathepsin L, whereas the converse situation applies with SmCL2. To characterize their catalytic subsites and substrate specificities, we have now developed three-dimensional (3D) homology models of SmCL1 and SmCL2 using the structure of cruzipain and cathepsin L. Eisenberg analysis of the 3D models revealed self-compatibility scores of 90.1 and 96.1 out of a possible score of 97.6 for SmCL1 and SmCL2, respectively, verifying the accuracy and utility of the models. Substrate preferences of recombinant SmCL1 and SmCL2 at positions P3, P2, and P1 conformed to the substrate specificity predicted by the models. In particular, SmCL1 and SmCL2 both exhibited high affinity (k(cat)/K(m)) for substrates with hydrophobic residues at P2 including Z-Leu-Arg-NHMec (773.4 and 548.5 mM(-1) s(-1), respectively), Boc-Val-Leu-Lys-NHMec (116.8 and 306.5 mM(-1) s(-1)), and Z-Phe-Arg-NHMec (38.9 and 113.4 mM(-1) s(-1)). SmCL1 exhibited only a low affinity for the cathepsin B diagnostic substrate Z-Arg-Arg-NHMec while SmCL2 failed to cleave this substrate. The substrate specificities of SmCL1 and SmCL2 were clearly differentiated with H-Leu-Val-Tyr-NHMec and Suc-Leu-Tyr-NHMec since SmCL1 cleaved both efficiently (k(cat)/K(m) values of 51.9 and 41.1 mM(-1) s(-1), respectively), whereas SmCL2 cleaved neither. The 3D models revealed that this difference in specificity was due to restrictions imposed on the S3 subsite of SmCL2 as a result of insertion of two amino acids vicinal to residue 60.

Functional expression of dipeptidyl peptidase I (Cathepsin C) of the oriental blood fluke Schistosoma japonicum in Trichoplusia ni insect cells.

Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel-nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS-PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH(2)-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues -180 to -175, NH(2)-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH(2)-terminal sequence, LDXNQLY, corresponding to residues -73 to -67 of the proregion peptide and thus were generated by removal of 126 residues from the NH(2)-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37 degrees C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k(cat)/K(m)) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM(-)1 s(-1), respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) High Five cells.

Juvenile Fasciola hepatica are resistant to killing in vitro by free radicals compared with larvae of Schistosoma mansoni.

Free radicals have previously been shown to kill the immature stages of the trematode, Schistosoma mansoni but their effect on newly excysted juvenile (NEJ) flukes of Fasciola hepatica has not been established. Using acetaldehyde and xanthine oxidase to chemically generate reactive oxygen intermediates (ROI), up to 61% of NEJ were killed but only when exposed to high levels of ROI. At low concentrations of acetaldehyde and xanthine oxidase as sources of reactive oxygen intermediates, only 6-29% of NEJ were killed compared with 70-92% of schistosomula. Incubation with lipopolysaccharide (LPS)-stimulated rat peritoneal lavage cells (PLCs) killed only 7-15% of NEJ whereas 78-87% of schistosomula were killed under the same conditions by a mechanism dependent on the production of reactive nitrogen intermediates. Relative to immature and adult parasites, NEJ expressed 2.5-20-fold lower levels of superoxide dismutase and glutathione S-transferase but no catalase activity was detected. Incubation of NEJ with inhibitors of peroxidases and glutathione metabolism increased the mean killing of NEJ by LPS-stimulated rat PLCs to 40-75%. These results demonstrate that, in comparison to schistosomula of S. mansoni, NEJ of F. hepatica are relatively resistant to killing by free radicals and this resistance could, in part, be due to the activity of oxidant scavenger enzymes of NEJ.