BACKGROUND: Avian schistosomes, the causative agents of human cercarial dermatitis (or swimmer's itch), die in mammals but the mechanisms responsible for parasite elimination are unknown. Here we examined the role of reactive nitrogen species, nitric oxide (NO) and peroxynitrite, in the immune response of mice experimentally infected with Trichobilharzia regenti, a model species of avian schistosomes remarkable for its neuropathogenicity. METHODS: Inducible NO synthase (iNOS) was localized by immunohistochemistry in the skin and the spinal cord of mice infected by T. regenti. The impact of iNOS inhibition by aminoguanidine on parasite burden and growth was then evaluated in vivo. The vulnerability of T. regenti schistosomula to NO and peroxynitrite was assessed in vitro by viability assays and electron microscopy. Additionally, the effect of NO on the activity of T. regenti peptidases was tested using a fluorogenic substrate. RESULTS: iNOS was detected around the parasites in the epidermis 8 h post-infection and also in the spinal cord 3 days post-infection (dpi). Inhibition of iNOS resulted in slower parasite growth 3 dpi, but the opposite effect was observed 7 dpi. At the latter time point, moderately increased parasite burden was also noticed in the spinal cord. In vitro, NO did not impair the parasites, but inhibited the activity of T. regenti cathepsins B1.1 and B2, the peptidases essential for parasite migration and digestion. Peroxynitrite severely damaged the surface tegument of the parasites and decreased their viability in vitro, but rather did not participate in parasite clearance in vivo. CONCLUSIONS: Reactive nitrogen species, specifically NO, do not directly kill T. regenti in mice. NO promotes the parasite growth soon after penetration (3 dpi), but prevents it later (7 dpi) when also suspends the parasite migration in the CNS. NO-related disruption of the parasite proteolytic machinery is partly responsible for this effect.

• Keywords
• 3-Nitrotyrosine, Cathepsin B, Nitric oxide, Nitric oxide synthase, Peroxynitrite, Schistosomatidae, Trichobilharzia,
• MeSH
• Central Nervous System parasitology   MeSH
• Guanidines pharmacology   MeSH
• Trematode Infections drug therapy   MeSH
• Skin parasitology   MeSH
• Peroxynitrous Acid pharmacology   MeSH
• Humans MeSH
• Spinal Cord parasitology   MeSH
• Mice MeSH
• Nitric Oxide pharmacology   MeSH
• Peptide Hydrolases drug effects   metabolism   MeSH
• Helminth Proteins drug effects   metabolism   MeSH
• Birds parasitology   MeSH
• Schistosoma drug effects   growth & development   pathogenicity   MeSH
• Schistosomatidae drug effects   growth & development   pathogenicity   MeSH
• Schistosomiasis drug therapy   MeSH
• Nitric Oxide Synthase drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Guanidines MeSH
• Peroxynitrous Acid MeSH
• Nitric Oxide MeSH
• pimagedine MeSH Browser
• Peptide Hydrolases MeSH
• Helminth Proteins MeSH
• Nitric Oxide Synthase MeSH

Schistosomula (the post-infective stages) of the neurotropic schistosome Trichobilharzia regenti possess multiple isoforms of cathepsin B1 peptidase (TrCB1.1-TrCB1.6) with involvement in nutrient digestion. The comparison of substrate preferences of TrCB1.1 and TrCB1.4 showed that TrCB1.4 had a very narrow substrate specificity and after processing it was less effective toward protein substrates when compared to TrCB1.1. Self-processing of both isoforms could be facilitated by sulfated polysaccharides due to a specific binding motif in the pro-sequence. Trans-activation by heterologous enzymes was also successfully employed. Expression profiling revealed a high level of transcription of genes encoding the enzymatically inactive paralogs TrCB1.5 and TrCB1.6. The transcription level of TrCB1.6 was comparable with that of TrCB1.1 and TrCB1.2, the most abundant active isoforms. Recombinant TrCB1.6wt, a wild type paralog with a Cys29-to-Gly substitution in the active site that renders the enzyme inactive, was processed by the active TrCB1 forms and by an asparaginyl endopeptidase. Although TrCB1.6wt lacked hydrolytic activity, endopeptidase, but not dipeptidase, activity could be restored by mutating Gly29 to Cys29. The lack of exopeptidase activity may be due to other mutations, such as His110-to-Asn in the occluding loop and Asp224-to-Gly in the main body of the mature TrCB1.6, which do not occur in the active isoforms TrCB1.1 and TrCB1.4 with exopeptidase activity. The catalytically active enzymes and the inactive TrCB1.6 paralog formed complexes with chicken cystatin, thus supporting experimentally the hypothesis that inactive paralogs could potentially regulate the activity of the active forms or protect them from being inhibited by host inhibitors. The effect on cell viability and nitric oxide production by selected immune cells observed for TrCB1.1 was not confirmed for TrCB1.6. We show here that the active isoforms of TrCB1 have different affinities for peptide substrates thereby facilitating diversity in protein-derived nutrition for the parasite. The inactive paralogs are unexpectedly highly expressed and one of them retains the ability to bind cystatins, likely due to specific mutations in the occluding loop and the enzyme body. This suggests a role in sequestration of inhibitors and protection of active cysteine peptidases.

• Keywords
• cathepsin B, cystatin, helminth, occluding loop, peptidase, processing, schistosome, substrate specificity,
• MeSH
• Astrocytes metabolism   MeSH
• Cystatins metabolism   MeSH
• Hydrolysis MeSH
• Isoenzymes metabolism   MeSH
• Cathepsin B chemistry   genetics   metabolism   MeSH
• Macrophages metabolism   MeSH
• Mice MeSH
• Nitric Oxide metabolism   MeSH
• Enzyme Precursors metabolism   MeSH
• Proteolysis MeSH
• RAW 264.7 Cells MeSH
• Recombinant Proteins metabolism   MeSH
• Schistosomatidae enzymology   pathogenicity   MeSH
• Amino Acid Substitution MeSH
• Substrate Specificity MeSH
• Protein Binding MeSH
• Cell Survival MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• cystatin, egg-white MeSH Browser
• Cystatins MeSH
• Isoenzymes MeSH
• Cathepsin B MeSH
• Nitric Oxide MeSH
• Enzyme Precursors MeSH
• Recombinant Proteins MeSH

Antibody trapping is a recently described strategy for immune evasion observed in the intestinal trematode Echinostoma caproni, which may aid to avoiding the host humoral response, thus facilitating parasite survival in the presence of high levels of local-specific antibodies. Parasite-derived peptidases carry out the degradation of trapped antibodies, being essential for this mechanism. Herein, we show that cathepsin-like cysteine endopeptidases are active in the excretory/secretory products (ESPs) of E. caproni and play an important role in the context of antibody trapping. Cysteine endopeptidase activity was detected in the ESPs of E. caproni adults. The affinity probe DCG-04 distinguished a cysteine peptidase band in ESPs, which was specifically recognized by an anti-cathepsin L heterologous antibody. The same antibody localized this protein in the gut and syncytial tegument of adult worms. Studies with cultured parasites showed that in vivo-bound antibodies are removed from the parasite surface in the absence of peptidase inhibitors, while addition of cathepsin L inhibitor prevented their degradation. These results indicate that cathepsin L-like peptidases are involved in the degradation of surface-trapped antibodies and suggest that cysteine peptidases are not only crucial for tissue-invading trematodes, but they can be equally relevant at the parasite-host interface in gut-dwelling flukes.

• Keywords
• Antibody, Cathepsin, Cysteine peptidase, Echinostoma caproni, Immune evasion, Trematode,
• MeSH
• Cysteine Endopeptidases metabolism   MeSH
• Echinostoma immunology   metabolism   MeSH
• Echinostomiasis immunology   parasitology   MeSH
• Immune Evasion immunology   MeSH
• Cathepsin L antagonists & inhibitors   MeSH
• Proteolysis MeSH
• Antibodies, Protozoan immunology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cysteine Endopeptidases MeSH
• Cathepsin L MeSH
• Antibodies, Protozoan MeSH

BACKGROUND: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. METHODS: Sequences of cathepsins L and B (EnCL and EnCB) were mined from E. nipponicum transcriptome and analysed bioinformatically. Genes encoding two EnCLs and one EnCB were cloned and recombinant proteins produced in vitro. The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Antibodies and specific RNA probes were employed for localisation of the enzymes/transcripts in tissues of E. nipponicum adults. RESULTS: Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I. They were localised in the haematin cells of the worm's digestive tract and in gut lumen. The EnCB showed similarity with cathepsin B2 of Schistosoma mansoni. It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. CONCLUSIONS: To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms. The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the (partially extracellular?) digestion of host blood proteins. High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis.

• Keywords
• Blood digestion, Cathepsin, Cysteine peptidase, Diplozoidae, Eudiplozoon nipponicum, Fish parasite, Haematophagy, Monogenea, Protease, S2 subsite,
• MeSH
• Gastrointestinal Tract parasitology   MeSH
• Hydrolysis MeSH
• Host-Parasite Interactions MeSH
• Carps parasitology   MeSH
• Cathepsin B chemistry   genetics   isolation & purification   metabolism   MeSH
• Cathepsin L chemistry   genetics   isolation & purification   metabolism   MeSH
• Proteolysis MeSH
• Recombinant Proteins analysis   genetics   isolation & purification   MeSH
• Gene Expression Profiling MeSH
• Trematoda enzymology   genetics   MeSH
• Introduced Species MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cathepsin B MeSH
• Cathepsin L MeSH
• Recombinant Proteins MeSH

BACKGROUND: Helminth neuroinfections represent a serious health problem, but host immune mechanisms in the nervous tissue often remain undiscovered. This study aims at in vitro characterization of the response of murine astrocytes and microglia exposed to Trichobilharzia regenti which is a neuropathogenic schistosome migrating through the central nervous system of vertebrate hosts. Trichobilharzia regenti infects birds and mammals in which it may cause severe neuromotor impairment. This study was focused on astrocytes and microglia as these are immunocompetent cells of the nervous tissue and their activation was recently observed in T. regenti-infected mice. RESULTS: Primary astrocytes and microglia were exposed to several stimulants of T. regenti origin. Living schistosomulum-like stages caused increased secretion of IL-6 in astrocyte cultures, but no changes in nitric oxide (NO) production were noticed. Nevertheless, elevated parasite mortality was observed in these cultures. Soluble fraction of the homogenate from schistosomulum-like stages stimulated NO production by both astrocytes and microglia, and IL-6 and TNF-α secretion in astrocyte cultures. Similarly, recombinant cathepsins B1.1 and B2 triggered IL-6 and TNF-α release in astrocyte and microglia cultures, and NO production in astrocyte cultures. Stimulants had no effect on production of anti-inflammatory cytokines IL-10 or TGF-β1. CONCLUSIONS: Both astrocytes and microglia are capable of production of NO and proinflammatory cytokines IL-6 and TNF-α following in vitro exposure to various stimulants of T. regenti origin. Astrocytes might be involved in triggering the tissue inflammation in the early phase of T. regenti infection and are proposed to participate in destruction of migrating schistosomula. However, NO is not the major factor responsible for parasite damage. Both astrocytes and microglia can be responsible for the nervous tissue pathology and maintaining the ongoing inflammation since they are a source of NO and proinflammatory cytokines which are released after exposure to parasite antigens.

• Keywords
• Anti-inflammatory cytokines, Astrocytes, Avian schistosome, Cathepsin B, Microglia, Neuroinfection, Nitric oxide, Proinflammatory cytokines, Trichobilharzia regenti,
• MeSH
• Astrocytes immunology   parasitology   MeSH
• Interleukin-6 metabolism   MeSH
• Cells, Cultured MeSH
• Mice MeSH
• Neuroglia immunology   parasitology   MeSH
• Nitric Oxide metabolism   MeSH
• Schistosomatidae immunology   MeSH
• Tumor Necrosis Factor-alpha metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• interleukin-6, mouse MeSH Browser
• Interleukin-6 MeSH
• Nitric Oxide MeSH
• Tumor Necrosis Factor-alpha MeSH

To date, most molecular investigations of schistosomatids have focused principally on blood flukes (schistosomes) of humans. Despite the clinical importance of cercarial dermatitis in humans caused by Trichobilharzia regenti and the serious neuropathologic disease that this parasite causes in its permissive avian hosts and accidental mammalian hosts, almost nothing is known about the molecular aspects of how this fluke invades its hosts, migrates in host tissues and how it interacts with its hosts' immune system. Here, we explored selected aspects using a transcriptomic-bioinformatic approach. To do this, we sequenced, assembled and annotated the transcriptome representing two consecutive life stages (cercariae and schistosomula) of T. regenti involved in the first phases of infection of the avian host. We identified key biological and metabolic pathways specific to each of these two developmental stages and also undertook comparative analyses using data available for taxonomically related blood flukes of the genus Schistosoma. Detailed comparative analyses revealed the unique involvement of carbohydrate metabolism, translation and amino acid metabolism, and calcium in T. regenti cercariae during their invasion and in growth and development, as well as the roles of cell adhesion molecules, microaerobic metabolism (citrate cycle and oxidative phosphorylation), peptidases (cathepsins) and other histolytic and lysozomal proteins in schistosomula during their particular migration in neural tissues of the avian host. In conclusion, the present transcriptomic exploration provides new and significant insights into the molecular biology of T. regenti, which should underpin future genomic and proteomic investigations of T. regenti and, importantly, provides a useful starting point for a range of comparative studies of schistosomatids and other trematodes.

• MeSH
• Adaptation, Biological * MeSH
• Host-Pathogen Interactions * MeSH
• Ducks parasitology   MeSH
• Metabolic Networks and Pathways genetics   MeSH
• Molecular Sequence Data MeSH
• Schistosomatidae genetics   growth & development   MeSH
• Sequence Analysis, DNA MeSH
• Life Cycle Stages MeSH
• Gene Expression Profiling * MeSH
• Computational Biology * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Bird schistosomes, besides being responsible for bird schistosomiasis, are known as causative agents of cercarial dermatitis. Cercarial dermatitis develops after repeated contact with cercariae, mainly of the genus Trichobilharzia, and was described as a type I, immediate hypersensitivity response, followed by a late phase reaction. The immune response is Th2 polarized. Primary infection leads to an inflammatory reaction that is insufficient to eliminate the schistosomes and schistosomula may continue its migration through the body of avian as well as mammalian hosts. However, reinfections of experimental mice revealed an immune reaction leading to destruction of the majority of schistosomula in the skin. Infection with the nasal schistosome Trichobilharzia regenti probably represents a higher health risk than infections with visceral schistosomes. After the skin penetration by the cercariae, parasites migrate via the peripheral nerves, spinal cord to the brain, and terminate their life cycle in the nasal mucosa of waterfowl where they lay eggs. T. regenti can also get over skin barrier and migrate to CNS of experimental mice. During heavy infections, neuroinfections of both birds and mammals lead to the development of a cellular immune response and axonal damage in the vicinity of the schistosomulum. Such infections are manifest by neuromotor disorders.

• Publication type
• Journal Article MeSH

Schistosomiasis caused by a parasitic blood fluke of the genus Schistosoma afflicts over 200 million people worldwide. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated peptidase that digests host blood proteins as a source of nutrients. It is under investigation as a drug target. To further this goal, we report three crystal structures of SmCB1 complexed with peptidomimetic inhibitors as follows: the epoxide CA074 at 1.3 Å resolution and the vinyl sulfones K11017 and K11777 at 1.8 and 2.5 Å resolutions, respectively. Interactions of the inhibitors with the subsites of the active-site cleft were evaluated by quantum chemical calculations. These data and inhibition profiling with a panel of vinyl sulfone derivatives identify key binding interactions and provide insight into the specificity of SmCB1 inhibition. Furthermore, hydrolysis profiling of SmCB1 using synthetic peptides and the natural substrate hemoglobin revealed that carboxydipeptidase activity predominates over endopeptidolysis, thereby demonstrating the contribution of the occluding loop that restricts access to the active-site cleft. Critically, the severity of phenotypes induced in the parasite by vinyl sulfone inhibitors correlated with enzyme inhibition, providing support that SmCB1 is a valuable drug target. The present structure and inhibitor interaction data provide a footing for the rational design of anti-schistosomal inhibitors.

• MeSH
• Hemoglobins chemistry   MeSH
• Hydrolysis drug effects   MeSH
• Protease Inhibitors chemistry   MeSH
• Cathepsin D antagonists & inhibitors   chemistry   genetics   MeSH
• Crystallography, X-Ray MeSH
• Drug Delivery Systems * MeSH
• Humans MeSH
• Peptidomimetics chemistry   MeSH
• Peptides chemistry   MeSH
• Helminth Proteins antagonists & inhibitors   chemistry   genetics   MeSH
• Schistosoma mansoni enzymology   genetics   MeSH
• Schistosomiasis mansoni drug therapy   enzymology   genetics   MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Hemoglobins MeSH
• Protease Inhibitors MeSH
• Cathepsin D MeSH
• Peptidomimetics MeSH
• Peptides MeSH
• Helminth Proteins MeSH

The neurotropic bird schistosome Trichobilharzia regenti possesses papain-like cysteine peptidases which have also been shown to be crucial enzymes in various developmental stages of the related human parasites Schistosoma spp. In this paper, we present data obtained by real-time polymerase chain reaction on the temporal distribution of transcripts of two cathepsins in different developmental stages of T. regenti: cathepsin B1 originally described from the gut lumen of schistosomula with presumptive role in nutrient digestion and cathepsin B2 originally found in penetration glands of cercariae with probable involvement in invasion of the final host. In spite of their mutual resemblance at the sequence level, the mRNA expression profiles clearly show distinct expression of cathepsins B1 and B2 during the development from eggs to cercariae. In the case of both cathepsins, the highest level of transcription was detected in intravertebrate stages. Putative functions of cathepsins B1 and B2 in schistosome developmental stages are discussed.

• MeSH
• Snails MeSH
• Isoenzymes genetics   metabolism   MeSH
• Ducks MeSH
• Cathepsin B genetics   metabolism   MeSH
• Helminth Proteins genetics   metabolism   MeSH
• Schistosomatidae enzymology   genetics   growth & development   MeSH
• Life Cycle Stages MeSH
• Gene Expression Profiling * MeSH
• Gene Expression Regulation, Developmental * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Isoenzymes MeSH
• Cathepsin B MeSH
• Helminth Proteins MeSH

A transcriptional product of a gene encoding cathepsin B-like peptidase in the bird schistosome Trichobilharzia regenti was identified and cloned. The enzyme was named TrCB2 due to its 77% sequence similarity to cathepsin B2 from the important human parasite Schistosoma mansoni. The zymogen was expressed in the methylotropic yeast Pichia pastoris; procathepsin B2 underwent self-processing in yeast media. The peptidolytic activity of the recombinant enzyme was characterised using synthetic fluorogenic peptide substrates at optimal pH 6.0. Functional studies using different specific inhibitors proved the typical cathepsin B-like nature of the enzyme. The S(2) subsite specificity profile of recombinant TrCB2 was obtained. Using monospecific antibodies against the recombinant enzyme, the presence of cathepsin B2 was confirmed in extracts from cercariae (infective stage) and schistosomula (early post-cercarial stage) of T. regenti on Western blots. Also, cross-reactivity was observed between T. regenti and S. mansoni cathepsins B2 in extracts of cercariae, schistosomula or adults. In T. regenti, the antisera localised the enzyme to post-acetabular penetration glands of cercariae implying an important role in the penetration of host skin. The ability of recombinant TrCB2 to degrade skin, serum and nervous tissue proteins was evident. Elastinolytic activity suggests that the enzyme might functionally substitute the histolytic role of the serine class elastase known from S. mansoni and Schistosoma haematobium but not found in Schistosoma japonicum or in bird schistosomes.

• MeSH
• Cysteine biosynthesis   MeSH
• Cysteine Proteases biosynthesis   genetics   physiology   MeSH
• Snails MeSH
• Ducks MeSH
• Cathepsin B biosynthesis   MeSH
• Rabbits MeSH
• Turkeys MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Pichia MeSH
• Schistosoma mansoni enzymology   MeSH
• Schistosoma enzymology   MeSH
• Amino Acid Sequence MeSH
• Cattle MeSH
• Substrate Specificity MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Humans MeSH
• Mice MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cysteine MeSH
• Cysteine Proteases MeSH
• Cathepsin B MeSH