20.500.12556/RUL-156089 Mehanizem delovanja in sodelovanja treh ključnih virulenčnih dejavnikov bakterije Listeria monocytogenes Mechanism of action and sinergy between three main Listeria monocytogenes virulence factors Listeria monocytogenes (Lm) je znotrajcelični bakterijski povzročitelj listerioze, potencialno smrtonosne okužbe, ki se prenaša s hrano. Trije glavni virulenčni dejavniki Lm so listeriolizin O (LLO) in dve fosfolipazi: širokospecifična fosfolipaza C (LmPC-PLC) in fosfatidilinozitol-specifična fosfolipaza C (LmPI-PLC). Glavni poudarek raziskav te doktorske naloge je bila funkcijska in strukturna analiza LmPC-PLC in njenih interakcij z LLO. Določili smo kristalno zgradbo LmPC-PLC in z dodatnimi biokemijskimi in biofizikalnimi eksperimenti smo pokazali dejavnike, ki regulirajo delovanje LmPC-PLC. Kristalna struktura razkriva, da je LmPC-PLC razvila več strukturnih posebnosti za uravnavanje svoje aktivnosti, vključno s položajem stranske skupine N-terminalnega triptofana (W1), strukturno plastičnim aktivnim mestom, od Zn2+ odvisno aktivnostjo in težnjo po tvorbi neaktivnih oligomerov. V nadaljevanju smo ugotovili aktivacijo LmPC-PLC s holesterolom, kar nakazuje na usmerjeno delovanje na gostiteljeve membrane. Opisali smo specifično inhibicijo LmPC-PLC z dodatkom njenega propetida in trans, kar odpira pot za razvoj specifičnih inhibitorjev, v namene zdravljenja okužb s sevi Lm, ki so odporni na antibiotike. Proučevali smo tudi sinergijo med LmPC-PLC in LLO ter ugotovili sinergistični vpliv LmPC-PLC na vezavo LLO na membrane in njegovo premabilizacijsko aktivnost. Ravno tako smo pokazali, da predinkubacija lipidnih membran z LmPC-PLC vpliva na morfologijo oligomerov, ki jih tvori LLO. Ta raziskava prinaša pomemben vpogled v strukturo in funkcijo LmPC-PLC, ki ima potencial kot terapevtska tarča pri zdravljenju listerioze, ter odkriva nove mehanizme sinergije med LmPC-PLC in LLO, kar znatno prispeva k razumevanju patofiziologije Lm. Listeria monocytogenes (Lm) is the intracellular bacterial agent of listeriosis, a potentially fatal foodborne infection. The three main virulence factors of Lm are listeriolysin O (LLO) and two phospholipases: broad-range phospholipase C (LmPC-PLC) and phosphatidylinositol-specific phospholipase C (LmPI-PLC). The main research focus of this PhD thesis was the functional and structural analysis of LmPC-PLC and its interactions with LLO. We have determined the crystal structure of LmPC-PLC and through additional biochemical and biophysical experiments we have shown the factors that regulate the function of LmPC-PLC. The crystal structure reveals that LmPC-PLC has evolved several structural features to regulate its activity, including the position of the N-terminal tryptophan side group (W1), a structurally plastic active site, Zn2+-dependent activity, and a tendency to form inactive oligomers. Next, we found the activation of LmPC-PLC by cholesterol, which suggests a directed action on the host membranes. We described the specific inhibition of LmPC-PLC by the addition of its propetide in trans, which opens the way for the development of specific inhibitors for the treatment of infections with antibiotic-resistant strains of Lm. We also studied the synergy between LmPC-PLC and LLO and found a synergistic effect of LmPC-PLC on the binding of LLO to membranes and its premobilization activity. We also showed that preincubation of lipid membranes with LmPC-PLC affects the morphology of oligomers formed by LLO. This research provides important insight into the structure and function of LmPC-PLC, which has potential as a therapeutic target in the treatment of listeriosis, and uncovers new mechanisms of synergy between LmPC-PLC and LLO, which significantly contributes to the understanding of the pathophysiology of Lm. interakcije membranskih proteinov z membranami listerijske fosfolipaze C listeriolizin O makromolekularna kristalografija presevna elektronska mikroskopija pod krio pogoji strukturne prilagoditve Listeria monocytogenes interactions of membrane proteins with membranes listerial phospholipases C listeriolysin O macromolecular crystallography CryoEM structural adaptations Listeria monocytogenes true false false [N. Petrišič] Slovenski jezik Angleški jezik Doktorsko delo/naloga 2024-05-08 07:15:44 2024-05-08 07:15:53 2024-05-09 08:39:10 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 66.095.261:66.065.5:543.442.3(043.3) 236517 194839299 39348.pdf 39348.pdf 1 659CD03494957FA4A0DE271FCABB11AB 3540e4296cbfc4e8454a5d4d9ca2816ff2a4cd2658628ba5ce8e67ea2121eadb 0d4d1029-0cfa-11ef-a389-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=183322 Biotehniška fakulteta 0 0 0