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Laboratory cycles of schistosomes, breeding of intermediate snail hosts and their ecological requirements
Nečasová, Monika ; Mikeš, Libor (advisor) ; Horák, Petr (referee)
The Schistosomatidae family is an important group of blood flukes, including serious human and livestock animal pathogens. With regard to reproduction, they are gonochoristic parasites. Their life cycle has two stages - asexual reproduction in an intermediary host (fresh water and sea gastropods) and sexual reproduction in the definitive host (mammals and birds). With regard to their pathogenicity at the definitive hosts, the blood fluke life cycle is abundantly maintained in laboratories. To preserve the whole cycle, the intermediary host gastropods are very important, They can include, without limitation, representatives of the Lymnaeidae family. However, their sensitivity to parasitism by blood flukes varies significantly by species. Floats were classified into several genera according to various parameters in the past. The systematics was most frequently based on anatomic and morphological differences. However, the most precise taxonomies are based on molecular data and mutual phylogenetic relationships of Lymnaeidaegastropods remain a subject of research. It is a fresh water family of gastropods with specific ecological and nutritional requirements. Summary information about their ecology estimate that the float gastropods are highly sensitive to the presence of heavy metals in water, such as...
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Excretory-secretory proteins of schistosome intravertebrate stages in context of their biology
Konečný, Lukáš ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Schistosomatidae is a family of blood-dwelling trematodes which cause serious disease in humans and warm blooded animals. Human schistosomiasis affects over 258 million people worldwide. The fundamental pathological manifestations of the disease are not caused by adult worms, but by responses to antigens released from eggs, which remain trapped in the tissues. All stages of these parasites release excretory-secretory products, which mostly comprise of proteins which are, to a certain extent, species- and stage-specific. These products have many functions that play roles in successful transmission of the parasite and its interactions with the host. Various approaches to the collection and analysis of these molecules do not always accurately reflect the secretory processes in vivo, because they always employ in vitro techniques. The research of these secreted molecules discovered many mechanisms, through which the parasite successfully modulates and evades host immune system. Despite the fact, that many of these molecules were identified as potential vaccine candidates, still no sufficiently effective vaccine has been developed. With great technological advances used for the identification of these proteins, this area is becoming more and more topical. This review focuses on composition of...
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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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Cathepsins L of Diplostomum pseudospathaceum cercariae
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Hartmann, David (referee)
This study is focused on cercarial cysteine peptidases of the trematode Diplostomum pseudospathaceum. It follows previous research which confirmed the presence of a 24kDa cysteine peptidase in cercariae biochemically and by mass spectrometry. It was postulated, that the function of this peptidase is histolytic, when cercariae penetrate the tissues. During an attempt to purify this peptidase and characterize its peptidolytic activity, it was found out that the cercarial homogenate containsmore different peptidases varying in their pI. Tests of peptidolytic activity and inhibition have shown that these peptidases are cathepsin L-like. They are active over a broad spectrum of pH with optima of activities in weakly acidicor neutral pH. Using degenerate primers based on conserved motifs of cysteine pepridases, partial sequences of three genes for cathepsin L of D. pseudospataceum (DpCL1, 2 a 3) were obtained. Then the complete sequences of DpCL2 and 3 genes and partial sequence (without 5'end) of DpCL1 were obtained by RACE PCR. To confirm function of these peptidases we tried to immunolocalize them. We assumed that they are localized in penetration glands. Preliminary results suggested that some of the cathepsins could be also localized in the gut of cercariae. For more detailed biochemical...
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Orientation of helminths during host seeking
Vojtová, Terezie ; Mikeš, Libor (advisor) ; Horák, Petr (referee)
The parasitic helminths are often forced in the environment to search for their host quickly in order to continue the ontogenetic development, or else to finish it. To search for and indentify the host, they use various types of receptors which allow them to provoke a taxes corresponding to the oncoming stimulus and to react regarding the current situation. In respect to the concept relevant to the helminths, this work concerns trematodes, whose freely movable stadia are miracidium and cercacia, and then monogeneans which are viparous or oviparous with oncomiracidium larva, and nematodes during which the third larva stage moves. The representatives of the groups of Myxozoans and Ciliophorea are also mentionned, since they use similar mechanisms to look for hosts as helminths. This thesis includes the summary of the existing knowledge related to the orientation of helminths in the environment. On the basis of the data that is already known, it is possible to compare which mechanisms are used for host-seeking by particular species, and according to which signals from the surroundings they most often orientate themselves. Powered by TCPDF (www.tcpdf.org)
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Morphological and functional variability of secretory glands in cercariae of chosen trematode groups
Krčmářová, Veronika ; Bulantová, Jana (advisor) ; Mikeš, Libor (referee)
Trematodes are characterized by their complex life cycles that include definitive hosts and variable number of intermediate hosts. Transfer of the parasite from the first intermediate host to the other is usually realized by larval stage called cercaria. After finishing of their development within the first intermediate host, morphology and fate of these larval stages vary according to the way of infection of the next host. Some cercariae actively penetrate directly to their definitive hosts trough their body surface, other encystate in the outer environment where they wait in a form of metacercariae to be ingested by definitive host. Both of these ways can be combinated and cercariae encystate inside second intermediate host after they actively penetrate them. Exceptionally, cercariae do not leave the sporocyst in which they were developing inside the first intermediate host. They encyst there waiting for ingestion by the definitive host. Various types of secretory glands have been developed in cercariae for successful direct infection of next hosts, survival of parasite in adverse conditions of outer environment or for transformation of one larval stage to subsequent one. Variability in morphology and function of these secretory glands in cercariae is closely connected with differences in life...
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Orientation of helminths during host seeking
Vojtová, Terezie ; Mikeš, Libor (advisor) ; Horák, Petr (referee)
Helminths are often forced in the environment to search for their host quickly in order to continue the ontogenetic development, or else to finish it. To search for and indentify the host, they use various types of receptors which allow them to provoke a taxes corresponding to the oncoming stimulus and to react to the presence of the host. This thesis includes the summary of the existing knowledge related to the orientation of helminths, the comparison of the mechanisms used by the searching of a host and signals in the environment. In respect to the concept relevant to the helminths, this work concerns trematodes, whose freely movable stadia are miracidium and cercaria, and then monogeneans which are viparous or oviparous with oncomiracidium larva, and nematodes with the third infective larva stage. The representatives of fish parasites of Myxozoans and Ciliophorea are also mentionned, since they use similar mechanisms to look for hosts as helminths. 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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Cathepsin L by parasites - occurrence, features, functions
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Cathepsines L are lysosomal cysteine endopeptidases with an universal function in protein catabolism. This work discusses present knowledge about their characteristics in the context of their specific function in parasites. Features and function differences are described in detail on molecular level. The emphasis is on the biochemical properties with resultant use of these enzymes. Cathepsines L of kinetoplastida, aplikomplexa, entamoeba and helmints (focused on Fasciola spp and Schistosoma spp) are each discussed in appropriate chapters. Key words: hydrolase, protease, cysteine peptidase, cathepsin L, lysosome, parasite
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Peptidases of monogeneans of the family Diplozoidae
Jedličková, Lucie ; Mikeš, Libor (advisor) ; Dvořák, Jan (referee)
The blood processing mechanisms in monogeneans of the subclass Polyopisthocotylea are known from ultrastructural and histochemical analyses only. In contrast to other blood- feeding parasites, just few biochemical and molecular analyses have been done on digestive enzymes in monogeneans. Therefore, we focused on the biochemical and molecular characterization of hydrolytic enzymes (peptidases) in the hematophagous species Paradiplozoon bliccae and Eudiplozoon nipponicum. The presence of the cysteine class peptidases, mainly cathepsin L, in excretory- secretory products and soluble protein extracts of P. bliccae and E. nipponicum we found. Detection was carried out using fluorogenic substrates, specific inhibitors and the labelled probe DCG-04. On the gels / membranes after electrophoresis / blotting we detected bands of approximately size of 35 kDa in the case of both species and 24 kDa for E. nipponicum. Soluble protein extracts of worms were separated by 2D gel electrophoresis and relevant spots around 35 kDa (P. bliccae) and around 25 ˗ 35 kDa (E. nipponicum) were confirmed by mass spectrometry as cathepsins L. Using degenerate primers based on the conserved motifs of cysteine class peptidases, a partial sequence of cathepsin L gene from E. nipponicum was obtained. Furthermore, 3'RACE PCR method...
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