Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni

BACKGROUND: Schistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths.

METHODS: We have cloned one gene sequence from S. mansoni, Smp_147730 (SmKI-1) which is coded for single domain Kunitz type protease inhibitor, E. coli-expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1.

RESULTS: SmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.

CONCLUSIONS: We have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.

Haem uptake is essential for egg production in the haematophagous blood fluke of humans, Schistosoma mansoni

Schistosomes ingest host erythrocytes, liberating large quantities of haem. Despite its toxicity, haem is an essential factor for numerous biological reactions, and may be an important iron source for these helminths. We used a fluorescence haem analogue, palladium mesoporphyrin, to investigate pathways of haem acquisition, and showed that palladium mesoporphyrin accumulates in the vitellaria (eggshell precursor glands) and ovary of female Schistosoma mansoni. Furthermore, incubation of adult females in 10-100 μm cyclosporin A (IC50 = 2.3 μm) inhibits the uptake of palladium mesoporphyrin to these tissues, with tenfold reductions in fluorescence intensity of the ovary. In vitro exposure to cyclosporin A resulted in significant perturbation of egg production, reducing egg output from 34 eggs per female to 5.7 eggs per female over the incubation period, and retardation of egg development. We characterized a S. mansoni homologue of the haem-responsive genes of Caenorhabditis elegans. The gene (Smhrg-1) encodes a protein with a molecular weight of approximately 17 kDa. SmHRG-1 was able to rescue growth in haem transport-deficient HEM1Δ yeast. Transcriptional suppression of Smhrg-1 in adult S. mansoni worms resulted in significant delay in egg maturation, with 47% of eggs from transcriptionally suppressed worms being identified as immature compared with only 27% of eggs laid by control worms treated with firefly luciferase. Our findings indicate the presence of transmembrane haem transporters in schistosomes, with a high abundance of these molecules being present in tissues involved in oogenesis.

Antibody Signatures Reflect Different Disease Pathologies in Patients With Schistosomiasis Due to Schistosoma japonicum

Infection with Schistosoma japonicum causes high levels of pathology that is predominantly determined by the cellular and humoral response of the host. However, the specific antibody response that arises during the development of disease is largely undescribed in Asian schistosomiasis-endemic populations. A schistosome protein microarray was used to compare the antibody profiles of subjects with acute infection, with early or advanced disease associated with severe pathology, with chronic infection, and subjects exposed but stool negative for S. japonicum eggs to the antibody profiles of nonexposed controls. Twenty-five immunodominant antigens were identified, including vaccine candidates, tetraspanin-related proteins, transporter molecules, and unannotated proteins. Additionally, individuals with severe pathology had a limited specific antibody response, suggesting that individuals with mild disease may use a broad and strong antibody response, particularly against surface-exposed proteins, to control pathology and/or infection. Our study has identified specific antigens that can discriminate between S. japonicum-exposed groups with different pathologies and may also allow the host to control disease pathology and provide resistance to parasite infection.

Multiplex real-time PCR monitoring of intestinal helminths in humans reveals widespread polyparasitism in Northern Samar, the Philippines

The global socioeconomic importance of helminth parasitic disease is underpinned by the considerable clinical impact on millions of people. While helminth polyparasitism is considered common in the Philippines, little has been done to survey its extent in endemic communities. High morphological similarity of eggs between related species complicates conventional microscopic diagnostic methods which are known to lack sensitivity, particularly in low intensity infections. Multiplex quantitative PCR diagnostic methods can provide rapid, simultaneous identification of multiple helminth species from a single stool sample. We describe a multiplex assay for the differentiation of Ascaris lumbricoides, Necator americanus, Ancylostoma, Taenia saginata and Taenia solium, building on our previously published findings for Schistosoma japonicum. Of 545 human faecal samples examined, 46.6% were positive for at least three different parasite species. High prevalences of S. japonicum (90.64%), A. lumbricoides (58.17%), T. saginata (42.57%) and A. duodenale (48.07%) were recorded. Neither T. solium nor N. americanus were found to be present. The utility of molecular diagnostic methods for monitoring helminth parasite prevalence provides new information on the extent of polyparasitism in the Philippines municipality of Palapag. These methods and findings have potential global implications for the monitoring of neglected tropical diseases and control measures.

Gaining biological perspectives from schistosome genomes

Characterization of the genomic basis underlying schistosome biology is an important strategy for the development of future treatments and interventions. Genomic sequence is now available for the three major clinically relevant schistosome species, Schistosoma mansoni, S. japonicum and S. haematobium, and this information represents an invaluable resource for the future control of human schistosomiasis. The identification of a biologically important, but distinct from the host, schistosome gene product is the ultimate goal for many research groups. While the initial elucidation of the genome of an organism is critical for most biological research, continued improvement or curation of the genome construction should be an ongoing priority. In this review we will discuss prominent recent findings utilizing a systems approach to schistosome biology, as well as the increased use of interference RNA (RNAi). Both of these research strategies are aiming to place parasite genes into a more meaningful biological perspective.

Characterisation of a secretory serine protease inhibitor (SjB6) from Schistosoma japonicum

BACKGROUND: Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential physiological roles in many organisms. In pathogens, serpins are thought to have evolved specifically to limit host immune responses by interfering with the host immune-stimulatory signals. Serpins are less well characterised in parasitic helminths, although some are thought to be involved in mechanisms associated with host immune modulation. In this study, we cloned and partially characterised a secretory serpin from Schistosoma japonicum termed SjB6, these findings provide the basis for possible functional roles.

METHODS: SjB6 gene was identified through database mining of our previously published microarray data, cloned and detailed sequence and structural analysis and comparative modelling carried out using various bioinformatics and proteomics tools. Gene transcriptional profiling was determined by real-time PCR and the expression of native protein determined by immunoblotting. An immunological profile of the recombinant protein produced in insect cells was determined by ELISA.

RESULTS: SjB6 contains an open reading frame of 1160 base pairs that encodes a protein of 387 amino acid residues. Detailed sequence analysis, comparative modelling and structural-based alignment revealed that SjB6 contains the essential structural motifs and consensus secondary structures typical of inhibitory serpins. The presence of an N-terminal signal sequence indicated that SjB6 is a secretory protein. Real-time data indicated that SjB6 is expressed exclusively in the intra-mammalian stage of the parasite life cycle with its highest expression levels in the egg stage (p < 0.0001). The native protein is approximately 60 kDa in size and recombinant SjB6 (rSjB6) was recognised strongly by sera from rats experimentally infected with S. japonicum.

CONCLUSIONS: The significantly high expression of SjB6 in schistosome eggs, when compared to other life cycle stages, suggests a possible association with disease pathology, while the strong reactivity of sera from experimentally infected rats against rSjB6 suggests that native SjB6 is released into host tissue and induces an immune response. This study presents a comprehensive demonstration of sequence and structural-based analysis of a secretory serpin from a trematode and suggests SjB6 may be associated with important functional roles in S. japonicum, particularly in parasite modulation of the host microenvironment.

Functional characterization of SjB10, an intracellular serpin from Schistosoma japonicum

Serine protease inhibitors (serpin) play essential roles in many organisms. Mammalian serpins regulate the blood coagulation, fibrinolysis, inflammation and complement activation pathways. In parasitic helminths, serpins are less well characterized, but may also be involved in evasion of the host immune response. In this study, a Schistosoma japonicum serpin (SjB10), containing a 1212 bp open reading frame (ORF), was cloned, expressed and functionally characterized. Sequence analysis, comparative modelling and structural-based alignment revealed that SjB10 contains the essential structural motifs and consensus secondary structures of inhibitory serpins. Transcriptional profiling demonstrated that SjB10 is expressed in adult males, schistosomula and eggs but particularly in the cercariae, suggesting a possible role in cercarial penetration of mammalian host skin. Recombinant SjB10 (rSjB10) inhibited pancreatic elastase (PE) in a dose-dependent manner. rSjB10 was recognized strongly by experimentally infected rat sera indicating that native SjB10 is released into host tissue and induces an immune response. By immunochemistry, SjB10 localized in the S. japonicum adult foregut and extra-embryonic layer of the egg. This study provides a comprehensive demonstration of sequence and structural-based analysis of a functional S. japonicum serpin. Furthermore, our findings suggest that SjB10 may be associated with important functional roles in S. japonicum particularly in host-parasite interactions.

Transcriptional profiling of the oesophageal gland region of male worms of Schistosoma mansoni

The intestinal tract of schistosomes opens at the mouth and leads into the foregut or oesophageal region that is lined with syncytium continuous with the apical cytoplasm of the tegument. The oesophagus is surrounded by a specialised gland, the oesophageal gland. This gland releases materials into the lumen of the oesophagus and the region is thought to initiate the lysis of erythrocytes and neutralisation of immune effectors of the host. The oesophageal region is present in the early invasive schistosomulum, a stage potentially targetable by anti-schistosome vaccines. We used a 44k oligonucleotide microarray to identify highly up-regulated genes in microdissected frozen sections of the oesophageal gland of male worms of S. mansoni. We show that 122 genes were up-regulated 2-fold or higher in the oesophageal gland compared with a whole male worm tissue control. The enriched genes included several associated with lipid metabolism and transmembrane transport as well as some micro-exon genes. Since the oesophageal gland is important in the initiation of digestion and the fact that it develops early after invasion of the mammalian host, further study of selected highly up-regulated functionally important genes in this tissue may reveal new anti-schistosome intervention targets for schistosomiasis control.

Unexpected Activity of a Novel Kunitz-Type Inhibtior: Inhibition of Cysteine Proteases but not Serine Proteases

Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity towards serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4 nM - 27 nM). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pull-down experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2 and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15/Arg15) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nM). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu15 in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity towards FhCL1, FhCL2 and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host.

Fasciola hepatica surface tegument: glycoproteins at the interface of parasite and host

Fasciola hepatica, commonly known as liver fluke, is a trematode which causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterisation of FhTeg glycosylation using lectin microarrays to characterise carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. While some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components which could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine.

Low efficacy of Mebendazole Against Hookworm in Vietman: Two Randomized Controlled Trials

Abstract. Vietnam is participating in a global de-worming effort that aims to treat 650 million school children regularly by 2010. The treatment used in Vietnam is single dose oral mebendazole (Phardazone®) 500 mg. We tested the efficacy of single dose mebendazole 500 mg in the therapy of hookworm infection in a randomized double-blind placebo-controlled trial among 271 Vietnamese schoolchildren. The treatment efficacy of single dose mebendazole in children did not differ significantly from placebo, with a reduction in mean eggs per gram of feces relative to placebo of 31% (95% CI - �9 to 56%, P = 0.1). In light of these findings we then carried out a similar randomized trial comparing triple dose mebendazole, single dose albendazole, and triple dose albendazole against placebo in 209 adults in the same area. The estimated reduction in mean post-treatment eggs per gram of feces relative to placebo was 63% (95% CI 30 - 81%) for triple mebendazole, 75% (47 - 88%) for single albendazole, and 88% (58Ã - 97%) for triple albendazole. Our results suggest that single dose oral mebendazole has low efficacy against hookworm infection in Vietnam, and that it should be replaced by albendazole. These findings are of major public health relevance given the opportunity costs of treating entire populations with ineffective therapies. We recommend that efficacy of anti-helminth therapies is pilot tested before implementation of national gut worm control programs.

Comparing concentration Methods: parasitrap® versus Kato-Katz for studying the prevalence of Helminths in Bengo province, Angola.

Background: Helminth intestinal parasitoses are responsible for high levels of child mortality and morbidity. Hence, the capacity to diagnose these parasitoses and consequently ensure due treatment represents a factor of great importance. Objectives: The main objective of this study involves comparing two methods of concentration, parasitrap and Kato-Katz, for the diagnosis of intestinal parasitoses in faecal samples. Methods: Sample processing made recourse to two different concentration Methods: the commercial parasitrap® method and the Kato-Katz method. Results: We correspondingly collected a total of 610 stool samples from pre-school and school age children. The results demonstrate the incidence of helminth parasites in 32.8% or 32.3% of the sample collected depending on whether the concentration method applied was either the parasitrap method or the Kato-Katz method. We detected a relatively high percentage of samples testing positive for two or more species of helminth parasites. We would highlight that in searching for larvae the Kato-Katz method does not prove as appropriate as the parasitrap method. Conclusion: Both techniques prove easily applicable even in field working conditions and returning mutually agreeing results. This study concludes in favour of the need for deworming programs and greater public awareness among the rural populations of Angola.

Neoechinorhynchus curemai (Acanthocephala : Neoechinorhynchidae) in Prochilodus lineatus (Osteichthyes : Prochilodontidae)from the Parana River, Brazil

This work evaluated, parasitic infections by Neoechinorhynchus curemai (Acanthocephala: Neoechinorhynchidae) in Prochilodus lineatus captured between August 2000 and August 2001 in the Parana River, Presidente Epitacio, São Paulo, Brazil. of 87 fishes examined, 59 were infected (25 males and 34 females). High mean intensities occurred in August 2000 (45.2, range 2-204), September 2000 (28.5, range 11-73), October 2000 (59.3, range 2-250) and February 2001 (27.3, range 3-73). There was no relationship of rainfall with mean intensity and prevalence. Males were more parasitized (P < 0.05) than females. This work contributes to the knowledge of helminth parasites of fish from a little studied region of the Parana River, showing diversity in their fauna when compared to other places in the same river. (c) 2005 Elsevier B.V. All rights reserved.