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Opredelitev lastnosti mutantov perfringolizina O s spremembami v holesterol-prepoznavnem delu
Sotlar, Inge (Author), Anderluh, Gregor (Mentor) More about this mentor... This link opens in a new window, Novinec, Marko (Co-mentor)

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Abstract
Toksin perfringolizin O (PFO) je pomemben virulenčni dejavnik grampozitivne bakterije Clostridium perfringens in ga uvrščamo v družino od holesterola odvisnih citolizinov, ki v membranah tarčnih celic tvorijo pore. Znano je, da se PFO veže na holesterol-vsebujoče membrane preko štirih zank v domeni 4 (D4). Ključni vpliv pri vezavi holesterola ima aminokislinski motiv T490-L491 v zanki L1. Točni molekularni mehanizem prepoznave membranskega holesterola še ni pojasnjen. Cilj magistrske naloge je bil opredeliti vpliv zamenjav aminokislinskih ostankov v zankah v D4 na aktivnost mutantov toksina PFO. Mutante PFO smo izrazili v baterijskem ekspresijskem sistemu E. coli in jih uspešno očistili z Ni-NTA afinitetno ter gelsko izključitveno kromatografijo. Potrdili smo, da imajo pravilno aminokislinsko zaporedje in molekulsko maso, z izjemo enega mutanta. Ugotovili smo, da se mutanti PFO z zamenjavami v paru T490-L491 vežejo na multilamelarne vezikle s 50 mol % holesterola. Vezave izbranih mutantov PFO na vezikle s 50 mol % holesteril acetata nismo opazili. Vpliv mutacij na aktivnost izbranih mutantov PFO smo opredelili z merjenjem hemolitične aktivnosti, kjer smo v skladu s predvidevanji ugotovili, da mutacije aminokislinskih ostankov različno vplivajo na aktivnost. Z metodo površinske plazmonske resonance (SPR) smo ugotovili, da se pri zamenjavi A401W afiniteta do holesterola poveča, medtem ko je zaradi aminokislinskih zamenjav v holesterol-prepoznavnem motivu (T490 in L491) afiniteta do holesterola manjša. Dokazali smo, da so se izbrani mutanti kljub zamenjavam v predlaganem holesterol-vezavnem motivu zmožni vezati na membrane s holesterolom.

Language:Slovenian
Keywords:od holesterola odvisni citolizini, perfringolizin O, lipidna membrana, holesterol
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2019
COBISS.SI-ID:1538509507 This link opens in a new window
Views:355
Downloads:147
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Secondary language

Language:English
Title:Characterisation of perfringolysin O mutants with substitutions in cholesterol-recognition site
Abstract:
The toxin perfringolysin O (PFO) is an important virulence factor of the Gram-positive bacterium Clostridium perfringens. It belongs to the family of cholesterol-dependent cytolysins, which form large pores in target cell membranes. PFO binds to cholesterol in the membrane by using four loops located in domain 4 (D4). The amino acid motif T490 L491, in loop L1, plays an important role in cholesterol binding. The exact mechanism of membrane cholesterol recognition at the molecular level is not yet completely understood. The aim of this thesis was to characterize the effects of amino acid substitutions in D4 loops on the activity of PFO mutants. PFO mutants were produced in the bacterial expression system Escherichia coli and were successfully purified with Ni-NTA affinity and size-exclusion chromatography. We demonstrated that the amino acid sequences and molecular masses of the PFO mutants were, with the exception of one mutant, as expected. Surprisingly, we observed binding of PFO mutants with substitutions in the pair T490-L491 to multilamellar vesicles with 50 mol % cholesterol. On the other hand, the tested PFO mutants did not bind to vesicles with 50 mol % cholesteryl acetate. Furthermore, we determined the effectiveness of the PFO mutants by measuring hemolytic activity. The amino acid substitutions considerably changed the PFO mutants activity. Using surface plasmon resonance (SPR), we found that the A401W substitution increased PFO affinity for cholesterol, whereas amino acid substitutions in the cholesterol recognition motif lowered the affinity for cholesterol. In conclusion, the chosen PFO mutants can bind to cholesterol containing membranes, despite the substitutions in the proposed cholesterol binding motif.

Keywords:cholesterol-dependent cytolysins, perfringolysin O, lipid membrane, cholesterol

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