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Characterisation of recombinant cathepsins B of the bird schistosome Trichobilharzia regenti
Dvořáková, Hana ; Mikeš, Libor (advisor) ; Dvořák, Jan (referee)
This study focuses on the recombinant cysteine peptidases - cathepsin B originating in the bird schistosome Trichobilharzia regenti that is unique across the whole family for its ability to migrate through the nerve tissue to the final localization. For invasion, migration, degradation of nutritional proteins and/or evasion of host immune responses, schistosome employs peptidases. This study follows the research done by researchers of Department of parasitology, Faculty of Natural Sciences, Charles University. The main goal of this study was to deepen the characteristics of recombinant cathepsins B originating in T. regenti. In T. regenti, two cysteine peptidases - cathepsins B1 (TrCB1) and B2 (TrCB2) - have been previously characterized. TrCB1 is located in the gut of schistosomula and involved in digestion. TrCB2 occurs in post-acetabular penetration glands of cercariae and probably facilitates penetration. The recombinant pro-cathepsin B (isoforms TrCB1.1, TrCB1.4 and also TrCB2) were expressed in Pichia pastoris yeast system. An attempt was made to produce in P. pastoris the recombinant isoform TrCB1.6, in which the active site cysteine is substituted by glycine. While TrCB2 underwent self-processing in the expression medium, TrCB1.1 and TrC1.4 zymogens were effectively activated only after the...
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Cathepsins B of the bird schistosome, Trichobilharzia regenti
Dolečková, Kateřina ; Horák, Petr (advisor) ; Grevelding, Christoph (referee) ; Horn, Martin (referee)
1. Overview Schistosomes have achieved first position among parasitic helminths, because some of them are the etiological agents of a serious human parasitic disease, schistosomiasis, which affects over 200 million people in tropical and subtropical countries (WHO, 2001). Other schistosomatids, such as the bird flukes of the genus Trichobilharzia, have also implications for human health. Although they can mature only in specific hosts (birds), their invasive larvae - cercariae - are able to penetrate also human skin due to chemical signals similar to those present on bird skin (Haas and van de Roemer 1998). Repeated infections result in an inflammatory reaction of the skin called cercarial dermatitis. Due to the increasing number of outbreaks all around the world, cercarial dermatitis is cons disease (Kolářová 2007idered as re-emerging ; Larsen et al. 2004). Among schistosomes, Trichobilharzia regenti is the only species described so far having a unique migration route within vertebrate hosts: after penetration of the skin, the invasive larvae enter peripheral nerves and continue via the spinal cord and central nervous system to the nasal cavity of birds, causing neuromotor disorders or paralyses of birds and even experimental mammals (Hrádková...
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Acid peptidases of schistosomes and haematophagous monogeneans
Dvořáková, Hana ; Mikeš, Libor (advisor) ; Horn, Martin (referee) ; Sojka, Daniel (referee)
Blood is a complex nutrient-rich mixture. No wonder that haematophagy has been adopted as a feeding strategy by many invertebrates, including many parasitic helminths. In general, processing of haemoglobin (and other blood proteins) in blood-feeding helminths relies on an evolutionary conserved network of cysteine and aspartic peptidases (e.g., cathepsins L, B and D). However, some helminth taxa have been neglected from this point of view - very little information has been available about the occurrence of these enzymes in haematophagous monogeneans. Therefore, the presented thesis focuses on the molecular and biochemical characteristics of peptidases that maybe potentially involved in blood processing by the monogenean Eudiplozoon nipponicum (Heteronchoinea, Diplozoidae), an ectoparasite inhabiting gills of common carp. We show that the most abundant haemoglobinolytic endopeptidase activities in soluble protein extracts and excretory/secretory products of E. nipponicum belong to the cysteine and aspartic classes, with cathepsin L-like activity predominating over cathepsin B-like activity and supplemented with cathepsin D-like activity (paper 1). Additionally, we found that E. nipponicum adults express a variety of cathepsins L with different structural characteristics and probably different...
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Bioactive molecules involved in blood processing by haematophagous monogeneans of the family Diplozoidae
Jedličková, Lucie
Monogeneans from the family Diplozoidae (subclass Heteronchoinea) are bloodfeeding ectoparasites inhabiting gills of common carp. Digestion of blood in diplozoids is an intracellular process taking place in gut cells within lysosomal cycle in the presence of parasite's peptidases. However, information about the blood digestion comes only from ultrastructural and histochemical analyses. Therefore, I have focused in this work on biochemical and molecular characteristics of bioactive molecules which may participate in blood processing by E. nipponicum adults, especially cysteine peptidases of cathepsin L- and B- types, aspartic peptidases of cathepsin D-type, and Kunitz-type inhibitors of serine peptidases. In homogenates and excretory/secretory (E/S) products of E. nipponicum adults, an activity of cysteine peptidases of cathepsins L-type dominated, followed by an activity of cathepsin D-like aspartic peptidases and a minor cathepsin B-like activity. Inhibitors of the abovementioned peptidase types completely blocked hemoglobinolytic activity in the samples. In the transcriptome of E. nipponicum adults, ten cathepsin L-coding transcripts were found and only one cathepsin B-coding transcript. Primary structures of the encoded enzymes were bioinformatically and phylogenetically compared. Two abundant...
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Bioactive molecules involved in blood processing by haematophagous monogeneans of the family Diplozoidae
Jedličková, Lucie
Monogeneans from the family Diplozoidae (subclass Heteronchoinea) are bloodfeeding ectoparasites inhabiting gills of common carp. Digestion of blood in diplozoids is an intracellular process taking place in gut cells within lysosomal cycle in the presence of parasite's peptidases. However, information about the blood digestion comes only from ultrastructural and histochemical analyses. Therefore, I have focused in this work on biochemical and molecular characteristics of bioactive molecules which may participate in blood processing by E. nipponicum adults, especially cysteine peptidases of cathepsin L- and B- types, aspartic peptidases of cathepsin D-type, and Kunitz-type inhibitors of serine peptidases. In homogenates and excretory/secretory (E/S) products of E. nipponicum adults, an activity of cysteine peptidases of cathepsins L-type dominated, followed by an activity of cathepsin D-like aspartic peptidases and a minor cathepsin B-like activity. Inhibitors of the abovementioned peptidase types completely blocked hemoglobinolytic activity in the samples. In the transcriptome of E. nipponicum adults, ten cathepsin L-coding transcripts were found and only one cathepsin B-coding transcript. Primary structures of the encoded enzymes were bioinformatically and phylogenetically compared. Two abundant...
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Bioactive molecules involved in blood processing by haematophagous monogeneans of the family Diplozoidae
Jedličková, Lucie ; Mikeš, Libor (advisor) ; Horn, Martin (referee) ; Sojka, Daniel (referee)
Monogeneans from the family Diplozoidae (subclass Heteronchoinea) are bloodfeeding ectoparasites inhabiting gills of common carp. Digestion of blood in diplozoids is an intracellular process taking place in gut cells within lysosomal cycle in the presence of parasite's peptidases. However, information about the blood digestion comes only from ultrastructural and histochemical analyses. Therefore, I have focused in this work on biochemical and molecular characteristics of bioactive molecules which may participate in blood processing by E. nipponicum adults, especially cysteine peptidases of cathepsin L- and B- types, aspartic peptidases of cathepsin D-type, and Kunitz-type inhibitors of serine peptidases. In homogenates and excretory/secretory (E/S) products of E. nipponicum adults, an activity of cysteine peptidases of cathepsins L-type dominated, followed by an activity of cathepsin D-like aspartic peptidases and a minor cathepsin B-like activity. Inhibitors of the abovementioned peptidase types completely blocked hemoglobinolytic activity in the samples. In the transcriptome of E. nipponicum adults, ten cathepsin L-coding transcripts were found and only one cathepsin B-coding transcript. Primary structures of the encoded enzymes were bioinformatically and phylogenetically compared. Two abundant...
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Proteolytic system of blood flukes of the genus Schistosoma
Bakardjieva, Marina ; Mareš, Michael (advisor) ; Dvořák, Jan (referee)
Blood flukes of the genus Schistosoma are parasitic trematodes causing a disease called schistosomiasis, which afflicts more than 200 million people in the tropics and subtropics. Adult schistosomes live in human blood vessels and feed on blood. Critical nutrients required for growth, development and reproduction of schistosomes are obtained from the major blood protein haemoglobin. Its digestion is mediated by the proteolytic arsenal of the schistosome digestive tract, which includes enzymes with complementary specificity belonging to the classes of cysteine and aspartic proteases, and metalloproteases. Proteolytic enzymes also play an important role in other processes, such as host penetration, tissue migration, immune evasion and modulation of inflammation. Here, serine and cysteine proteases importantly participate. The proteolytic system is essential for the viability of schistosomes and is a current topic of intense research focused on the development of new vaccines and chemotherapeutics for the treatment of schistosomiasis. Powered by TCPDF (www.tcpdf.org)
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Structural and functional analysis of cathepsin B1 from the blood fluke, Schistosoma mansoni
Jílková, Adéla ; Mareš, Michael (advisor) ; Obšil, Tomáš (referee) ; Mikeš, Libor (referee)
Schistosomiasis is a serious infectious disease that afflicts over 200 million people in tropical and subtropical regions. It is caused by Schistosoma blood flukes that live in human blood vessels and obtain nutrients from host hemoglobin, which is degraded by digestive proteases. Current therapy relies on a single drug and concern over resistance necessitates new drug development. In Schistosoma mansoni, cathepsin B1 (SmCB1) is a critical digestive protease that is a target molecule for therapeutic interventions. This thesis provides a comprehensive characterization of SmCB1 focused on structure-activity relationships and inhibitory regulation based on six crystal structures solved for SmCB1 molecular forms and complexes. SmCB1 is biosynthesized as an inactive zymogen in which the N-terminal propeptide operates as a natural intra-molecular inhibitor by blocking the active site. Detailed biochemical and structural analyses have identified a new and, so far, unique mechanism of SmCB1 zymogen activation through which the propeptide is proteolytically removed and the regulatory role of glycosaminoglycans in this process has been described. A study of SmCB1 proteolytic activity has revealed that the enzyme acts in two modes, as endopeptidase and exopeptidase, which makes it an efficient tool for host...
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Characterisation of recombinant cathepsins B of the bird schistosome Trichobilharzia regenti
Dvořáková, Hana ; Mikeš, Libor (advisor) ; Dvořák, Jan (referee)
This study focuses on the recombinant cysteine peptidases - cathepsin B originating in the bird schistosome Trichobilharzia regenti that is unique across the whole family for its ability to migrate through the nerve tissue to the final localization. For invasion, migration, degradation of nutritional proteins and/or evasion of host immune responses, schistosome employs peptidases. This study follows the research done by researchers of Department of parasitology, Faculty of Natural Sciences, Charles University. The main goal of this study was to deepen the characteristics of recombinant cathepsins B originating in T. regenti. In T. regenti, two cysteine peptidases - cathepsins B1 (TrCB1) and B2 (TrCB2) - have been previously characterized. TrCB1 is located in the gut of schistosomula and involved in digestion. TrCB2 occurs in post-acetabular penetration glands of cercariae and probably facilitates penetration. The recombinant pro-cathepsin B (isoforms TrCB1.1, TrCB1.4 and also TrCB2) were expressed in Pichia pastoris yeast system. An attempt was made to produce in P. pastoris the recombinant isoform TrCB1.6, in which the active site cysteine is substituted by glycine. While TrCB2 underwent self-processing in the expression medium, TrCB1.1 and TrC1.4 zymogens were effectively activated only after the...
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