Strukturna in funkcijska primerjava homologov širokospecifične fosfolipaze C iz bakterije Listeria monocytogenes ter karakterizacija njene prooblike

Adamek, Maksimiljan (author), Podobnik, Marjetka (mentor), Pavšič, Miha (co-mentor)

Nekatere bakterijske fosfolipaze C so pomembni virulentni dejavniki v patogenezi bakterijskih okužb. Tako je v primeru Listeria monocytogenes, ki je znotrajcelična patogena bakterija in povzročiteljica potencialno smrtonosne bolezni listerioze. V tem magistrskem delu smo primerjali homologne širokospecifične fosfolipaze C iz bakterij Listeria monocytogenes (PC PLCLm), Bacillus cereus (PC PLCBc) in Clostridium perfringens (PC-PLCCp) ter proučevali proobliko listerijske fosfolipaze C (proPC-PLCLm). Vse proteine smo izolirali rekombinantno s heterolognim izražanjem v bakteriji Escherichia coli ter očistili s hitin afinitetno kromatografijo in gelsko izključitveno kromatografijo. S spektroskopijo cirkularnega dikroizma smo pokazali, da imajo vsi proteini v večini α-vijačno zvitje in so zelo termostabilni. S kolorimetrično metodo smo analizirali aktivnosti encimov do substratov v obliki multilamelarnih lipidnih veziklov (MLV). Pri enotnem, fiziološko relevantnem reakcijskem pogoju (MLV iz 100 % POPC, pH 6,5, 50 µM ZnSO4, 1 mM CaCl2) je za posamezni homolog fosfolipazna aktivnost padala v vrsti: PC-PLCBc, PC-PLCCp, PC PLCLm. Pri PC-PLCLm in PC PLCCp sta bili fosfolipazna in sfingomielinazna aktivnost medsebojno primerljivi, medtem ko je bila sfingomielinazna aktivnost PC PLCBc za 60 % nižja od fosfolipazne. ProPC PLCLm je po pričakovanjih kazala nizko fosfolipazno aktivnost. Lipidna sestava MLV (razmerje POPC, sfingomielina in holesterola) je pomembno vplivala na encimsko aktivnost. Testi kosedimentacije so pokazali, da sta vezavi prooblike in zrele oblike PC PLCLm na MLV primerljivi. ProPC PLCLm se je s časom odcepljal propeptid. Analiza proteolizne stabilnosti z dolgotrajno inkubacijo je pokazala, da inkubacija v pufru s pH 4,5 bistveno upočasni proteolizno cepitev proPC PLCLm. Dodatek EDTA in enojne zamenjave aminokislinskih ostankov aktivnega mesta PC PLCLm niso bistveno povečali stabilnosti prooblike. Ugotovitve iz tega magistrskega dela predstavljajo dobro izhodišče za nadaljnjo primerjavo homolognih PC PLC in proučevanje prooblike PC PLCLm.

• Language: Slovenian
• Keywords: fosfolipaza C, Listeria monocytogenes, Bacillus cereus, Clostridium perfringens, prooblika
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-131191
• COBISS.SI-ID: 85700099
• Publication date in RUL: 23.09.2021

Secondary language

• Language: English
• Title: Structural and functional comparison of homologs of broad-range phospholipase C from Listeria monocytogenes and characterisation of its proform

Abstract

Some bacterial phospholipases C are important virulent factors in the pathogenesis of bacterial infections. This is also the case with Listeria monocytogenes, an intracellular pathogenic bacterium and a causative agent of the potentially life threatening disease, listeriosis. In this thesis, we have investigated the proform of listerial PC-PLCLm (proPC PLCLm) and homologous broad range phospholipases C from Listeria monocytogenes (PC-PLCLm), Bacillus cereus (PC PLCBc), and Clostridium perfringens (PC-PLCCp). All proteins were isolated recombinantly by heterologous expression in Escherichia coli, and purified by chitin affinity chromatography and size exclusion chromatography. Circular dichroism spectroscopy showed that all proteins have an α-helical structure and are highly thermostable. A colorimetric method was used to determine enzyme activities with multilamellar lipid vesicles (MLVs) used as substrates. At a single and physiologically relevant test condition (MLVs from 100 % POPC, pH 6.5, 50 µM ZnSO4, 1 mM CaCl2) PC PLCBc showed the highest fosfolipase activity, followed by PC PLCCp and PC PLCLm. PC-PLCLm and PC PLCCp had comparable sphingomyelinase and phospholipase activities, whereas the sphingomyelinase activity of PC-PLCBc was 60 % lower than the phospholipase activity. As expected, proPC-PLCLm had very low phospholipase activity. MLV lipid composition (ratio of POPC, sphingomyelin, and cholesterol) had an important effect on enzyme activity. Cosedimentation assays showed that the proform and the mature form of PC PLCLm had comparable MLV binding. Experiments with proPC PLCLm showed that the propeptide was cleaved of with time. Proteolytic stability analysis with long-term incubation showed that the propeptide cleavage was significantly slowed at pH 4.5. The addition of EDTA and single amino acid substitutions did not significantly improve the stability of proPC PLCLm. The results of this thesis provide a good basis for further comparison of homologous PC-PLCs and study of the proform of PC PLCLm.

• Keywords: phospholipase C, Listeria monocytogenes, Bacillus cereus, Clostridium perfringens, proform