SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

BackgroundSerine 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 findingsSmSP2 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/significanceThe 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

BIRD SCHISTOSOMES: development of schistosomula with focus on Trichobilharzia spp

5 ABSTRACT Schistosomulum is the first stage developing in definitive host body, affecting various body parts and in the case of bird schistosomes present in host tissues for longest period. The aims of the present thesis are to summarize recent knowledge of bird schistosomula migration, development and pathogenic impact on host tissues and complete the details for two model species (Trichobilharzia szidati and T. regenti) with different life strategy. The other aim was to introduce and test the method for in vitro cultivation of schistosomula. Schistosomulum is formed by transformation of cercaria in the host skin at the time of penetration. The process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. All this take place also under defined in vitro conditions. Transformed schistosomula migrate towards the target organ in host body. Depending on the species schistosomula migrate via the circulatory system or nervous tissues and the migration is directed either to intestinal or nasal area (visceral or nasal species, respectively). Specific migratory pattern for lung passage of T. szidati and migratory route of T. regenti through the nervous system, unique among schistosomes and including intra- and extra vascular location, are obligatory for..

BIRD SCHISTOSOMES: development of schistosomula with focus on Trichobilharzia spp

5 ABSTRACT Schistosomulum is the first stage developing in definitive host body, affecting various body parts and in the case of bird schistosomes present in host tissues for longest period. The aims of the present thesis are to summarize recent knowledge of bird schistosomula migration, development and pathogenic impact on host tissues and complete the details for two model species (Trichobilharzia szidati and T. regenti) with different life strategy. The other aim was to introduce and test the method for in vitro cultivation of schistosomula. Schistosomulum is formed by transformation of cercaria in the host skin at the time of penetration. The process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. All this take place also under defined in vitro conditions. Transformed schistosomula migrate towards the target organ in host body. Depending on the species schistosomula migrate via the circulatory system or nervous tissues and the migration is directed either to intestinal or nasal area (visceral or nasal species, respectively). Specific migratory pattern for lung passage of T. szidati and migratory route of T. regenti through the nervous system, unique among schistosomes and including intra- and extra vascular location, are obligatory for..

Vývoj schistosomul ptačích schistosom se zaměřením na rod Trichobilharzia

5 ABSTRACT Schistosomulum is the first stage developing in definitive host body, affecting various body parts and in the case of bird schistosomes present in host tissues for longest period. The aims of the present thesis are to summarize recent knowledge of bird schistosomula migration, development and pathogenic impact on host tissues and complete the details for two model species (Trichobilharzia szidati and T. regenti) with different life strategy. The other aim was to introduce and test the method for in vitro cultivation of schistosomula. Schistosomulum is formed by transformation of cercaria in the host skin at the time of penetration. The process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. All this take place also under defined in vitro conditions. Transformed schistosomula migrate towards the target organ in host body. Depending on the species schistosomula migrate via the circulatory system or nervous tissues and the migration is directed either to intestinal or nasal area (visceral or nasal species, respectively). Specific migratory pattern for lung passage of T. szidati and migratory route of T. regenti through the nervous system, unique among schistosomes and including intra- and extra vascular location, are obligatory for...{ ) I SEZNAMVLASTNiCHPUBLIKACi: cHANovA M, vuoNc s, HOMK P- (2007): Trichobilhunia szidatii the rung phase of migration within avian and mmmaliin hosts.Pamitol Res. 100(6): 1243- 1247(IFt.s) cHANovA M.' HoRAKP. (2007): Terminal phase ofbird schistosomiasiscaused by Tichobilhunia regezri (schistosomatidae) in ducks (Anu pktltrhynclros f. domstie). Folia pd6itol 54(2): 105 - 1070F r.o) cHANovA M, BULANToVAJ, MASLo p, HoRiK p (2009): In vito cultivation of erly shistosomula of nasal and visceral bird *histosomes (Trichobilhuziu spp., Schistommatidae). pmsitol Res 104(6):l44s - 14520F 1.5) zAviR V1/sledky pr6ce rozSiiuji dosavadni poaatky o biologii schistosomul ptaiich schistosom. Byly zji5tdni detailni infomace o rlivoji dvou modeloqich druhri rodu Trichobilhania repreantujicich viscer6lni a nm6lni schistosomy. Nejvi;amdjii vlsledky pr6ce: In vilro kultiryaee ' Schistosomuly trmsfomovmd in vivo \ziskmd z tkrini ptadiho hostitele) pieZivaji a dospivaji v podmir/rlieh in vitro v kultivadnim m6diu (RpMI 1620) s ptadimi erytrocyty pfi 37"C a 5o/oCO2. . RPMI 1620 medim se neosvEddilo pro in vitro tramfomrci a n6sledn51.vlvoj ptadich schistosom_ SCM 169, m6dim pro kultivaci lidsklch schistoso4 doplndni kachnimi erytrocl,ty a piisluinj'mi mtimykotiky a antibiotiky,je pro vjvoj rilich schistosomul vhodnd. ' Vlivojovd...Department of ParasitologyKatedra parazitologieFaculty of SciencePřírodovědecká fakult

Inactivation of Schistosoma Using Low-Temperature Plasma

The inactivation of Schistosoma mansoni cercariae and miracidia was achieved by exposure to plasma produced by the positive, negative, and axial negative corona discharges. The positive discharge appeared as the most effective, causing the death of cercariae and miracidia within 2–3 min of exposure. The negative discharge was less effective, and the axial discharge was ineffective. The water pre-activated (PAW) by the discharges showed similar efficiency, with the exception of the significantly effective PAW activated with axial discharge. These facts, together with the observation of various reactions among plasma-damaged schistosomes, suggest that the mechanisms of inactivation by different types of discharges are different

SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

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

Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis.

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

Substrate specificity and pH profile of SmSP2.

<p>(A) Activity of rSmSP2 was probed using a panel of peptidyl fluorogenic substrates used to assay trypsin-like and chymotrypsin/elastase-like serine proteases. Substrate hydrolysis was measured in a kinetic assay at pH 8.0. The mean values ± S.D. of three replicates are normalized to the maximum value. Amino acid residues at P1 and P2 positions are highlighted by the grey bar. (B) The pH profiles of rSmSP2 and native SmSP2 activity in extracts of adult worms. Activity was measured in a kinetic assay using the fluorogenic substrate P-F-R-AMC. The native activity (sensitive to the serine protease inhibitor Pefabloc SC) was measured in the presence of 10 μM E-64 and 1 mM EDTA to prevent undesired proteolysis of the substrate by cysteine proteases and metalloproteases, respectively. The mean values of three replicates, expressed as a percentage normalized to the highest value, are shown (standard deviation values are within 5% of the mean). (C) The pH stability of rSmSP2. Activity of rSmSP2 was measured at pH 8.0 in a kinetic assay as in (B) after incubation of the enzyme at pH 3 to 11 for different times. The mean values of three replicates, expressed as a percentage normalized to activity of non-incubated rSmSP2, are shown.</p

Sensitivity of rSmSP2 to protease inhibitors.

<p>Sensitivity of rSmSP2 to protease inhibitors.</p

Preparation of recombinant SmSP2 and identification of native SmSP2.

<p>(A) The recombinant protease domain of SmSP2 (rSmSP2) expressed in <i>P</i>. <i>pastoris</i> was resolved by SDS-PAGE and protein-stained or visualized by polyclonal anti-rSmSP2 IgG. For in-gel activity-based labeling, rSmSP2 was incubated with the fluorescent active site probe, BoRC, resolved by SDS-PAGE and visualized using a fluorescence scanner. The competitive labeling was performed with the serine protease inhibitor, Pefabloc SC. (B) Protein extracts of <i>S</i>. <i>mansoni</i> adult worms and their ESP were resolved by SDS-PAGE and visualized by the anti-rSmSP2 IgG.</p