Příprava mutantních variant cytotoxinu RtxA bakterie Kingella kingae pro studium membránové topologie

Lichvárová, Michaela

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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