Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

• MeSH
• kathepsin B * antagonisté a inhibitory   metabolismus   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• Schistosoma mansoni * enzymologie   účinky léků   MeSH
• schistosomicidy farmakologie   chemie   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Clostridial collagenases are essential biotechnological tissue dissociation agents owing to their ability to cleave different types of collagen. Standardization of collagenase-based protocols has been hampered by impurities in products manufactured from Clostridium histolyticum. To enhance the purification process, we produced recombinant collagenase classes G and H, taking advantage of the Escherichia coli expression system. The respective gene sequences were derived from C. histolyticum and modified by addition of a C-terminal polyhistidine tag. Harvested bacteria were lysed and the collagenase protein was affinity purified using a His-tag column. The purity, identity, integrity of the eluted collagenases G and H were determined by SDS electrophoresis and Western blot. The proteolytic activity of the collagenase G and H blend (rColGH) was determined by the standard FALGPA assay. The tissue dissociation activity was verified using a standardized method for isolation of rat pancreatic islets. Biocompatibility of the blend was validated by a standardized viability assay on the isolated islets. Two batches of rColGH were produced and compared to a commercially available collagenase. Based on our results, we conclude that rColGH is a functional and non-toxic novel recombinant collagenase worth further characterization and blend optimization in order to make it a competitive commercial product.

• MeSH
• Clostridium MeSH
• kolagenasy * MeSH
• krysa rodu rattus MeSH
• Langerhansovy ostrůvky * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Individual developmental stages of blood fluke Schistosoma mansoni excrete or secrete a different set of molecules. Here we describe optimized protocols for collection of excretory/secretory products (E/S products) from cercariae, schistosomula, adult worms, and eggs. These E/S products are essential for successful parasitism functioning at the host-parasite interface, enabling invasion into the host and contributing to the survival of the parasite by modulation of host physiology and immune responses. Collection of sufficient amounts of E/S products is required for detailed research of these processes.

• MeSH
• cerkárie fyziologie   MeSH
• játra parazitologie   MeSH
• myši MeSH
• odběr biologického vzorku metody   MeSH
• ovum fyziologie   MeSH
• Schistosoma mansoni růst a vývoj   metabolismus   MeSH
• stadia vývoje * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fasciolóza je parazitární onemocnění způsobované dvěma druhy motolic rodu Fasciola. Jedná se o jednu z nejvýznamnějších parazitóz hospodářských zvířat, vzácná není ani nákaza člověka. Masivní používání léčiva triklabendazolu vedlo k tvorbě rezistentních kmenů fasciol, které jsou v současnosti velmi rozšířené. Proto se věnuje mimořádné úsilí vývoji vakcíny. Proteolytický systém fasciol se účast-ní řady procesů nezbytných pro parazita, od líhnutí miracidií přes pronikání tělem hostitele a ovlivňování jeho fyziologických procesů až po trávení živin. Z tohoto důvodu jsou proteolytické enzymy atraktivním cílem pro vývoj vakcín proti fasciolóze. Tento článek pojednává o nejdůležitějších proteasách fasciol a jejich použití jako vakcinačních antigenů.

Fasciolosis is a parasitic infection caused by two species of flukes from the genus Fasciola. It is considered one of the most important diseases of livestock, but human infection is also common. Massive usage of the drug triclabendazole led to the development of resistant flukes, which are currently spread out in many countries. Hence there is much effort put into development of vaccines against fasciolosis. Proteolytic system of Fasciola plays crucial role in number of processes such as hatching, tissue invasion, nutrient intake or modulation of the host physiology. For that reason proteases of Fasciola are attractive targets for vaccine development. This paper summarizes current knowledge about the most important proteases from Fasciola hepatica and their vaccination potential.

• MeSH
• Fasciola hepatica * enzymologie   parazitologie   růst a vývoj   MeSH
• fasciolóza * patofyziologie   prevence a kontrola   MeSH
• hospodářská zvířata MeSH
• kathepsin B antagonisté a inhibitory   chemie   terapeutické užití   MeSH
• kathepsin L chemie   imunologie   terapeutické užití   MeSH
• vývoj vakcíny MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

BACKGROUND: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting. CONCLUSIONS/SIGNIFICANCE: The data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy.

• MeSH
• fibrinolýza účinky léků   MeSH
• hemokoagulace účinky léků   MeSH
• hemostatika antagonisté a inhibitory   MeSH
• krevní tlak účinky léků   MeSH
• molekulární modely MeSH
• plazminogen účinky léků   MeSH
• proteinové domény MeSH
• proteiny červů chemie   genetika   farmakologie   MeSH
• proteolýza účinky léků   MeSH
• rekombinantní proteiny MeSH
• Schistosoma mansoni enzymologie   MeSH
• schistosomiasis mansoni parazitologie   MeSH
• sekvence aminokyselin MeSH
• serinové endopeptidasy chemie   genetika   farmakologie   MeSH
• tkáňový aktivátor plazminogenu účinky léků   MeSH
• vazodilatace účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Schistosomiasis is one of a number of chronic helminth diseases of poverty that severely impact personal and societal well-being and productivity. Peptidases (proteases) are vital to successful parasitism, and can modulate host physiology and immunology. Interference of peptidase action by specific drugs or vaccines can be therapeutically beneficial. To date, research on peptidases in the schistosome parasite has focused on either the functional characterization of individual peptidases or their annotation as part of global genome or transcriptome studies. We were interested in functionally characterizing the complexity of peptidase activity operating at the host-parasite interface, therefore the excretory-secretory products of key developmental stages of Schistosoma mansoni that parasitize the human were examined. Using class specific peptidase inhibitors in combination with a multiplex substrate profiling assay, a number of unique activities derived from endo- and exo-peptidases were revealed in the excretory-secretory products of schistosomula (larval migratory worms), adults and eggs. The data highlight the complexity of the functional degradome for each developmental stage of this parasite and facilitate further enquiry to establish peptidase identity, physiological and immunological function, and utility as drug or vaccine candidates.

• MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• ovum metabolismus   MeSH
• proteasy metabolismus   sekrece   MeSH
• proteiny červů metabolismus   sekrece   MeSH
• proteolýza MeSH
• Schistosoma mansoni růst a vývoj   metabolismus   fyziologie   MeSH
• schistosomiasis mansoni parazitologie   MeSH
• sekvence aminokyselin MeSH
• stadia vývoje MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH