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Investigation of the acyltransferase RtxC interaction with the Kingella kingae toxin RtxA
Lichvárová, Michaela ; Osičková, Adriana (advisor) ; Černá, Věra (referee)
The bacterium Kingella kingae was first isolated in 1960 by microbiologist Elizabeth O. King and until recently, it was considered a rare cause of human disease. However, over the past 30 years, an increasing number of papers have shown that this bacterium is an important paediatric pathogen, mainly affecting children aged 6 months to 3 years, causing mainly septic arthritis, osteomyelitis, infective endocarditis, and bacteraemia. K. kingae displays a strong cytotoxic effect against a variety of host cell types, which is caused by the secreted cytolysin RtxA, a member of the RTX (Repeats in ToXin) family. RtxA binds to glycosylated structures of the host cell, subsequently inserts into its cytoplasmic membrane, and forms cation-selective pores, leading to disruption of ion homeostasis and lysis of the attacked cell. RtxA is produced as an inactive protoxin proRtxA. Its activation is mediated by the acyltransferase RtxC, which transfers acyl chains to conserved lysine residues K558 and K689 in the protoxin. It uses the acyl carrier protein ACP as the acyl donor. Currently, it is unclear how the RtxC, acyl-ACP, and proRtxA proteins interact with each other and which amino acid residues are responsible for these interactions. The aim of this thesis was to identify the residues responsible for these...
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Preparation of mutant variants of Kingella kingae RtxA cytotoxin for membrane topology research
Lichvárová, Michaela ; Osičková, Adriana (advisor) ; Malý, Petr (referee)
Kingella kingae is a facultative anaerobic, β-hemolytic, gram-negative bacterium. It has been shown, that K. kingae is an important cause of invasive infections in young children, especially between 6 to 36 months of age. The most common diseases caused by K. kingae are septic arthritis, osteomyelitis, bacteremia and infective endocarditis. The key virulence factor of K. kingae is the secreted RtxA toxin, which belongs to the RTX toxins family (Repeats in ToXin). These are divided into two categories, hemolysins and leukotoxins, based on the cellular specificity of their action. The broad specificity of the RtxA toxin indicates that RtxA can be classified as a cytolytic RTX hemolysin. RtxA molecules are inserted into the host cell membrane and form cation-selective membrane pores that trigger cation flux. This disrupts normal cell physiology and eventually leads to cell lysis. The aim of this bachelor thesis was to prepare mutant variants of the K. kingae RtxA cytotoxin with lysine substitutions in the pore-forming domain for future study of the membrane topology of the toxin using biotin binding to the lysine residues. In order to observe the topology of the RtxA toxin in the host cell membrane, the toxin must be able to insert to the cell membrane. Therefore, another objective was to determine...
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