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Structure-function relationship of Kingella kingae RtxA toxin.
Růžičková, Eliška ; Osička, Radim (advisor) ; Šulc, Miroslav (referee)
Kingella kingae is a pediatrically significant, facultative pathogen. It asymptomatically colonizes the oropharynx of young children, where it is a part of the normal microflora. However, if it penetrates the respiratory epithelial barrier and begins to spread throughout the body, it can cause serious infectious diseases. Thanks to today's advanced diagnostic methods, K. kingae is included among important human pathogens, and in pediatric patients, K. kingae is reported as a frequent cause of osteoarticular infections, such as osteomyelitis and septic arthritis, bacteremia, and endocarditis. The key virulence factor of this bacterium, the cytotoxin RtxA, belongs to the RTX (Repeats in ToXin) toxin family. This family of toxins shares several characteristic features: (i) the presence of a hydrophobic pore-forming domain in the N-terminal part of the molecule containing several predicted transmembrane α-helices (ii) the inactive protoxin is activated by different types of fatty acids bound to specific lysine residues in the acylated domain, (iii) the presence of nonapeptide repeat sequences, rich in glycine and aspartate residues, that are important for the binding of calcium ions, (iv) the presence of a C-terminal secretion signal that is recognized by the type I secretion system (T1SS), and (v)...
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Structure and function of RTX toxins of Gram-negative bacteria
Zhuk, Karyna ; Osička, Radim (advisor) ; Šulc, Miroslav (referee)
RTX toxins (Repeats in ToXin) are produced by Gram-negative bacteria, most of which are important human or animal pathogens. The polypeptide chain of each RTX toxin consists of four conserved regions. An N-terminal hydrophobic domain, which is important for insertion of the RTX toxin into the host cell membrane and pore formation. The hydrophobic domain is followed by an acylated segment containing conserved lysine residues, at which the toxin is acylated and thus activated. The C-terminal portion of each RTX toxin contains a repeat domain to which calcium ions bind. The C-terminus of the toxin contains a secretion signal that is recognized by the type I secretion system, which transports the toxin from the bacterial cytosol to the external environment. After secretion, RTX toxins interact with the cell surface via specific β2 integrins and/or glycosylated structures such as glycoproteins and gangliosides or membrane components such as sphingomyelins and cholesterol. Once bound to the cell, RTX toxin monomers insert into the membrane and oligomerize to form pores. The uncontrolled flow of ions through these pores can lead to disruption of bactericidal functions of myeloid phagocytes, stimulation or suppression of the release of pro-inflammatory cytokines, modulation of various signaling and...
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Proteomická a bioinformatická charakterizace N-terminálních sekvencí proteinů modifikovaných po importu do hydrogenosomu Trichomonas vaginalis.
Zákoucká, Eva ; Man, Petr (advisor) ; Šulc, Miroslav (referee)
Trichomonas vaginalis is a human pathogen causing trichomoniasis, one of the most common non-viral sexually transmitted diseases in both men and women. Trichomoniasis is currently treated with metronidazole, but the pathogen is known to develop resistance against this drug. However as the pathogen is eukaryotic, the targets for the pathogen elimination without seriously affecting the host are limited. Throughout the evolution Trichomonas vaginalis adapted to anaerobic environments by developing an alternative metabolism resulting in a reduced form of mitochondria named hydrogenosome. Hydrogenosomes lack genetic information, therefore all its proteins are nucleus-encoded and need to be transported inside the hydrogenosome using a targeting N-terminal presequence. The peptidase recognizing and cleaving those presequences at the entrance of the organelle, the hydrogenosomal processing peptidase (HPP), is unique for hydrogenosomes and therefore represents a potential drug target against the pathogen. In this work the HPP's substrate specificity towards the targeting presequences was investigated. To do so a proteomic analysis of the proteome of Trichomonas vaginalis hydrogenosomes was performed using a novel optimized protocol for N-terminal peptide sequencing. N-terminal peptides were captured using a...
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Nest construction
Jenšovský, Václav ; Kreisinger, Jakub (advisor) ; Šulc, Michal (referee)
The nest is in the life of birds important structure. It's a place where kept for posterity during the entire period of reproduction. For this to be nest offspring suitable place for development, must have a specific structure which is variable for each species. Variability, whether at the level of intraspecific and interspecific level and is influenced by several factors. These factors include thermal insulation properties, antipredatory properties, further property to protect the nest before (ecto) parasites, but also can design nest pose as a selectable character sexually. Each of those factors differently closer together, but often one factor may disadvantage second and thus reduce the success of rearing offspring. Key words: nest construction, thermal insulation properties, antipredatory properties, ectoparasites, sexual selection
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Analogues of IGF-1 for the study of interactions of the hormone with the receptors for IGF-1 and insulin
Macháčková, Kateřina ; Jiráček, Jiří (advisor) ; Obšilová, Veronika (referee) ; Šulc, Miroslav (referee)
Insulin/IGF system is a complex network of three similar hormones (insulin, IGF-1 and IGF-2) and their three similar receptors (IR-A, IR-B and IGF-1R,), which play important roles in maintaining basal energy homeostasis of the organism, in growth, development, life-span but also in development of diseases such as diabetes mellitus, cancer, acromegaly or Laron dwarfism. Despite structural similarities between family members, each member have its unique role in the system. Identification of structural determinants in insulin and IGFs that trigger their specific signalling pathways is important for rational drug design for safer treatment of diabetes or for more efficient combating of cancer or growth-related disorders. In this thesis, we focused on identification of such structural determinants in IGF-1. Comparison of our data with parallel studies with IGF-2 and insulin could give a more complex picture of the problem. First of all, we developed necessary methodologies for the preparation of IGF-1 analogues. We developed a new methodology for the total chemical synthesis of IGF-1 analogues based on the solid-phase synthesis of fragments and their ligation by a CuI -catalyzed cycloaddition of azides and alkynes. In parallel, we developed a procedure for a recombinant production of IGF- 1 and its...
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Kinetic properties of β-N-acetylhexosaminidase from tobacco plants
Valenta, Robert ; Ryšlavá, Helena (advisor) ; Šulc, Miroslav (referee)
β-N-acetylhexosaminidase from tobacco leaves (Nicotiana tabacum L.) was partially purified to final specific activity 1,72 µmol . min-1 . mg-1 using p-nitrofenyl-β-N- acetyl-D-glucosaminide as substrate. The enzyme exhibited one band after both isoelectric focusing and native electrophoresis. Molecular mass of native enzyme was determined by gel chromatography (MR 275000) and native electrophoresis (MR 285000). Isoelectric point pI 5.4 was determined by isoelectric focusing. Activity of β-N-acetylhexosaminidase was measured using substrates p-nitrofenyl-β-N-acetyl-D-galactosaminide, p-nitrofenyl-β- N-acetyl-D-glucosaminide, N,N'-diacetylchitobiose, p-nitrofenyl-N,N'- diacetylchitobioside and N,N',N''-triacetylchitotriose. For substrates N,N'- diacetylchitobiose, p-nitrofenyl-N,N'-diacetylchitobioside and N,N',N''-triacetylchitotriose an enzyme assay of β-N-acetylhexosaminidase using capillary zone electrophoresis was developed. Optimal pH and temperature of β-N-acetylhexosaminidase were determined with individual substrates, as well as products of hydrolysis. Activity of β-N- acetylhexosaminidase was highest using p-nitrofenyl-β-N-acetyl-D-glucosaminide as substrate and lowest using N,N',N''-triacetylchitotriose (35% in relative comparison). Maximum velocity and Michaelis constant of...
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