Interakcije listeriolizina O s holesterolom

Kozorog, Mirijam (author), Anderluh, Gregor (mentor), Plavec, Janez (comentor)

Patogeni organizmi so razvili številne mehanizme, da se izognejo imunskemu sistemu gostitelja in se v njegovih celicah nadalje razmnožujejo. V ta namen izločajo različne virulenčne dejavnike, ki preko interakcij s komponentami gostiteljskih celic omogočijo preživetje patogenov. Glavni virulenčni dejavnik bakterije Listeria monocytogenes, ki je živalski in človeški patogen, je 56,4 kDa protein listeriolizin O (LLO). LLO se veže na membrane, bogate s holesterolom, v njih tvori pore ter s tem listeriji omogoči pobeg iz kislega fagosoma in njeno nadaljnje širjenje v sosednje celice in tkiva gostitelja. V doktorskem delu smo na molekularnem nivoju preučevali vezavo LLO na holesterol ter interakcije proteina z membranami, kar v literaturi še ni podrobno pojasnjeno. V ta namen smo izrazili rekombinanten LLO z vsemi sedmimi triptofanskimi ostanki označenimi z izotopom 19F (19F-LLO). Ugotovili smo, da se LLO močno veže na sam holesterol v raztopini, vendar pa noben od preučevanih triptofanskih ostankov, ki so izpostavljeni na dnu vezavne domene LLO, v vezavo ni bil direktno udeležen. Nasprotno smo pri študijah z 19F NMR spektroskopijo v trdnem opazili spremembe v kemijskih premikih treh triptofanskih ostankov LLO proteina, ko je bil le-ta vezan na s holesterolom bogate fosfolipidne vezikle. Glede na literaturo in rezultate izvedenih testov hemolitične aktivnosti triptofanskih mutant sklepamo, da so omenjeni triptofanski ostanki vključeni v oligomerizacijo proteina LLO v procesu tvorbe pore (W189 in W489) oziroma v interakcije z membranami (W512). Nadalje smo s setom NMR eksperimentov v trdnem pokazali, da vezava LLO na s holesterolom bogate membrane različnih sestav ne poruši organizacije lipidnega dvosloja, se pa v prisotnosti bolj fluidnih membran poveča mobilnost fosfolipidnih glav ter fluidnost hidrofobnega dela fosfolipidnega dvosloja. Na takšnih membranah opazimo tudi večji delež vezanega proteina kot na bolj rigidnih membranah pri isti mejni molski koncentraciji holesterola, ki je še zadostna za vezavo LLO na membrane. Z določevanjem relaksacijskih hitrosti holesterola smo ugotovili, da na membrane vezan LLO močno interagira z membranskim holesterolom v okolici njegovih ogljikovih atomov C3 in C4. Doktorsko delo tako omogoča podrobnejši vpogled v interakcije toksina LLO s holesterolom in s s holesterolom bogatimi fosfolipidnimi dvosloji. Pokaže tudi učinkovitost uporabe tehnik aminokislinsko specifičnega izotopskega označevanja večjih proteinov, ki omogočajo interakcijske študije podobno velikih proteinov z lipidnimi molekulami in membranskimi sistemi na molekularnem nivoju s tehnikama NMR v raztopini in NMR v trdnem. Tekom naloge so bili razviti tudi modelni lipidni sistemi na osnovi arhejskih lipidov, ki so uporabni za raziskave interakcij proteinov z lipidnimi membranami.

• Language: Slovenian
• Keywords: listeriolizin O, Listeria monocytogenes, od holesterola odvisni citolizini, vezava na membrane, holesterol, jedrska magnetna resonanca (NMR)
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2019
• PID: 20.500.12556/RUL-106038
• COBISS.SI-ID: 298575616
• Publication date in RUL: 18.01.2019

Secondary language

• Language: English
• Title: Interactions of listeriolysin O with cholesterol

Abstract

Pathogens have developed a number of mechanisms to avoid the host's immune system and reproduce in its cells. In order to do so, various virulence factors that allow the survival of the pathogen are produced. The main virulence factor of animal in human pathogen bacterium Listeria monocytogenes is protein listeriolysin O (LLO) with a molecular mass of 56,4 kDa. LLO binds to cholesterol-rich membranes, where it forms pores and thus allows the listeria to escape from acidic phagosome. This directly enables the bacterial survival and its spread to neighbouring host cells and tissues. In the doctoral thesis we studied the binding of LLO to cholesterol and the interactions of the protein with membranes at the molecular level, which has not been explained yet in detail. For this purpose, we expressed a recombinant LLO protein with all seven tryptophan residues labelled with 19F isotope. The results showed that LLO binds strongly to the free cholesterol in the solution, but none of the studied tryptophan residues that are exposed at the bottom of the binding domain of LLO were directly involved in the binding. In contrast, chemical shifts changes in three tryptophan residues were observed with 19F solid-state NMR spectroscopy when LLO was bound to cholesterol-rich phospholipid vesicles. According to the literature and to the hemolytic activities of single tryptophan mutants we concluded that these tryptophan residues are involved in the oligomerization of LLO protein in the process of pore formation (W189 and W489) or in the interactions with membranes (W512). With a set of solid-state NMR experiments we further showed that LLO binding to cholesterol-rich membranes out of different compositions does not disrupts the lipid bilayer organization. However, LLO binding increases the mobility of phospholipid head groups and the fluidity of the hydrophobic core of the phospholipid bilayer in the case of more fluid membranes. On these membranes a higher portion of bound LLO is observed when compared to more rigid membranes with equal molar concentration of cholesterol that is still sufficient for LLO binding. Further, the cholesterol relaxation experiments revealed that the membrane-bound LLO interacts strongly with membrane cholesterol in vicinity of cholesterol’s C3 in C4 carbon atoms. Doctoral thesis thus provide a more detailed insight into the interactions of LLO with cholesterol in cholesterol-rich phospholipid bilayers. It also demonstrates the usefullness of aminoacid-specific labelling of larger proteins in membrane-protein interaction studies at the molecular level with the solution and solid-state NMR methods. Also new membrane systems out of archaeal lipids and cholesterol were developed that can be used also in the future for interaction studies of proteins with lipid membranes.

• Keywords: listeriolysin O, Listeria monocytogenes, cholesterol-dependent cytolysins, membrane-binding, cholesterol, nuclear magnetic resonance (NMR)