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Characterization of serine proteases of Schistosoma mansoni involved in interaction with host
Ulrychová, Lenka ; Horn, Martin (advisor) ; Schabussova, Irma (referee) ; Sojka, Daniel (referee)
Schistosomiasis is a serious parasitic disease, caused by blood flukes of the genus Schistosoma. It remains a global health problem in the 21st century, with more than 250 million people infected in 78 countries. Current therapy relies on the drugs praziquantel and oxamniquine, for which there are concerns of emerging drug resistance. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising targets for the development of new therapeutic strategies against schistosomiasis. This work focuses on the characterization of Schistosoma mansoni serine proteases (SmSPs) and the determination of their role in the interaction with the human host using a variety of genomic, bioinformatic, RNA- and protein-based techniques. First, the major types of proteolytic activities secreted by the blood-dwelling developmental stages of S. mansoni were classified using functional proteomics. The analysis revealed the complexity of proteolytic activities secreted by the schistosome life stages parasitizing the human host. All stages secreted significant serine protease activities, and consequently their genes were retrieved from the genome database and annotated. Localization in adult worms determined by fluorescence in situ RNA hybridization revealed complex expression...
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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 ; 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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Analysis of secretome from Trichobilharzia regenti cercariae and characterisation of selected peptidases
Konečný, Lukáš ; Kašný, Martin (advisor) ; Horn, Martin (referee)
(English): Trichobilharzia regenti is a neurotropic parasite of birds from the family Schistosomatidae. Cercariae, the invasive stages of these trematodes actively penetrate the host skin employing excretory- secretory products (ESPs), which contain proteolytic enzymes able to disrupt host tissues and thus reach the successful transmission. The most abundant secreted enzyme responsible for cercarial penetration of the human schistosome S. mansoni is a cercarial elastase. This serine peptidase is well known for the degradation of skin proteins such as elastin, keratin, collagen or laminin. However, the active expression of the orthologue of this enzyme has never been found in the genus Trichobilharzia. For this reason, it was firmly believed, that cercaria of T. regenti uses mainly cysteine peptidases for the invasion of the host, particularly cathepsins, which were repeatedly identified in this life stage. To strengthen this hypothesis, we incubated T. regenti cercariae in the apparatus with the excised duck skin stimulating the release of their glands' content. The collected ESPs were further analysed by shotgun mass-spectrometry and for the first time, the protein form of cercarial elastase was identified. Unfortunately, we failed to produce its active recombinant protein in yeast and bacterial...
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Proteolytic systems of the blood fluke (Schistosoma mansoni).
Fajtová, Pavla ; Horn, Martin (advisor) ; Bařinka, Cyril (referee) ; Sojka, Daniel (referee)
Schistosomiasis is a serious parasitic disease caused by blood flukes of the genus Schistosoma. It is a global health problem with more than 200 million people infected and 750 million people at risk. Current therapy relies on a single drug, praziquantel, for which there are concerns of emerging drug resistance. Proteases of schistosoma are promising target molecules for the development of new therapeutic strategies against schistosomiasis. This work focuses on the comprehensive characterization of proteolytic systems of Schistosoma mansoni and determination of their role in the interaction with the human host. First, the major proteolytic activities secreted by individual developmental stages of schistosoma that parasitize the human body were classified using functional proteomics. This analysis demonstrated their complex and specific distribution with predominant serine and cysteine proteases and metalloproteases. Second, tegumental and digestive proteases, namely prolyl oligopeptidase and cathepsins B, C and D, were identified by chemical genomics as suitable target molecules for therapeutic intervention. Prolyl oligopeptidase was biochemically characterized using a recombinant protein, its effective inhibitors were developed as templates for antischistosomal drugs, and a biological role of the...
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Calcium and calcium-dependent proteins in the biology of schistosomes
Bartoníček, Zikmund ; Mikeš, Libor (advisor) ; Horn, Martin (referee)
Blood flukes of genus the Schistosoma are blood dwelling parasites that affect over 200 million people causing seriously debilitating disease, schistosomiasis. Like in other life forms, calcium represents one of the key elements in schistosomes. Calcium affects egg hatching, penetration into the host, evasion of hosts immune system and other crucial aspects of Schistosoma life. It can regulate those processes in two ways; either directly, or through interaction with calcium-binding proteins. Those proteins are either expressed in every life stage of schistosomes or they can be stage-specific. It is those properties of calcium and calcium-dependent proteins, that make them a potent vaccine targets. The first pioneer in the calcium dependent protein based vaccines is soon to come to human trials. Until the efficient vaccine is developed, we are dependent purely on chemotherapy against schistosomiasis. At the moment the drug of first choice, praziquantel is used to treat those who suffer from schistosomiasis. Its mode of action is not entirely known, but is evidently directly linked to calcium homoeostasis of schistosomes. This thesis focuses on calcium and calcium-dependent proteins because of their role - either direct or indirect - in the stated processes. Also, increased understanding of calcium...
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Characterization of excretory-secretory proteins of liver fluke Fascioloides magna.
Beránková, Kateřina ; Kašný, Martin (advisor) ; Horn, Martin (referee)
Fascioloides magna (the giant liver fluke) originated from North America, is known in the Czech Republic since 1930s. This pathogenic fluke invades mostly cervids, but livestock too. Excretory-secretory products (ES products) contain number of esential biomolecules which are produced by excretory and secretory system of the fluke. These molecules play key role in many biological process during the life cycle not only of fascioloid flukes (e.g. migration in the host tissues, immune evasion and digestion). Due to their antigenic properties they could be also used in immunodiagnostics. Excretory-secretory proteins from adult Fascioloides magna and comparative related species Fasciola hepatica were purified and separated by the basic biochemical methods (1D, 2D electrophoresis, ion-exchange chromatography) and their activity was confirmed by specific (fluorogenic peptide) and nonspecific (gelatine) substrates. By using the mass spectrometry methods (MALDI TOF/TOF), the most abundant peptidolytically active proteins from ES products of F. magna were identified as cathepsin L (FmCL). Recombinant analog of FmCL was expressed in Pichia pastoris expression system. The peptidolytic activity was again confirmed using the synthetic fluorogenic substrates; the specifity of recombinant FmCL active site was...
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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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