Biofizikalna in strukturna analiza oligomerov listeriolizina O

Strgar, Mojca (author), Podobnik, Marjetka (mentor)

Listeria monocytogenes je znotrajcelična patogena bakterija, ki povzroča listeriozo. Eden izmed glavnih virulentnih dejavnikov, ki omogoča njeno širjenje po gostitelju, je porotvorni protein listeriolizin (LLO). Bakterija LLO izloča v obliki topnih monomerov, ki na s holesterolom bogatih membranah oligomerizirajo in pri tem tvorijo večje oligomerne komplekse različnih velikosti – proteinske loke in obroče. Ti oligomeri po vgreznjenju v membrane tvorijo funkcionalne pore. Namen magistrske naloge je bil poiskati metode oz. kombinacije metod s katerimi bi lahko sledili nastanku teh oligomerov v različnih modelnih membranskih sistemih (t. j. liposomih, eritrocitih). Uporabili smo NaDS-PAGE in agarozno elektroforezo, ultracentrifugiranje v saharoznem gostotnem gradientu ter presevno-elektronsko mikroskopijo z negativnim senčenjem. Vse te metode so potrdile, da LLO tvori heterogene oblike oligomerov v primerjavi s homolognim proteinom perfringolizinom O (PFO), kjer smo z uporabo enakih pristopov dobili bolj homogene oligomere, v večini v obliki sklenjenega obroča. Oligomere LLO, ki so nastali bodisi na liposomih ali na eritrocitih, smo ekstrahirali iz membran. Analiza frakcij z ultracentrifugiranjem v saharoznem gostotnem gradientu je pokazala, da je večina LLO oligomerov v obliki lokov in le zelo redki dosežejo obliko sklenjenega obroča. Uporabljeni protokol za pripravo teh oligomerov je omogočil dobro ločbo LLO oligomerov od ostalih eritrocitnih proteinov in lipidov, je pa preveč robusten za bolj natančno ločevanje LLO oligomerov po velikosti. Vendar pa so rezultati te študije zelo dobra podlaga za nadaljnje strukturne raziskave LLO oligomerizacije oziroma tvorbe por na višjem nivoju ločljivosti, kot je na primer krio-elektronska mikroskopija.

• Language: Slovenian
• Keywords: Listeria monocytogenes, listerioza, proteini, toksini, citolizini, listeriolizin O, oligomeri, pore, biokemijska analiza
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2019
• PID: 20.500.12556/RUL-106480
• COBISS.SI-ID: 5003599
• Publication date in RUL: 27.02.2019

Secondary language

• Language: English
• Title: Biophysical and structural analysis of lysterioysin O oligomeres

Abstract

Listeria monocytogenes is intracellular bacterial pathogen that causes listeriosis. One of the main virulent factors that enables spreading of the bacteria is pore-forming protein listeriolysin O (LLO). Bacteria secretes water-soluble monomers LLO. These monomeres bind to the cholesterol-rich membrane where they oligomerize into protein complexes of different sizes - protein arcs and circles. After membrane insertion these oligomeres form functional forms. The main purpose of this thesis was to find methods or combination of methods that will enable us to follow steps of oligomerization in different membrane modell systems (e. g. liposomes, erythrocytes). We used SDS-PAGE and agarose electrophoresis, ultracentrifugation in sucrose density gradient and transmission microscopy with negative staining. All methods confirmed that LLO forms more heterogenous oligomeres in comparison with homolog protein perfirngolysin O (PFO) which we used in the same methods as LLO and confirmed that PFO oligomeres are mainly circles. Oligomeres formed on membrane of liposomes or erythrocytes were extracted from membranes. Analyzing fractions from ultracentrifugation in sucrose density gradient showed that most of LLO oligomeres are in the form of arcs and that only few oligomeres form full circle. Protocol we used for preparation of these oligomeres allowed us good seperation of LLO oligomeres from other erythrocyte proteins and lipids, but it is too robust for precise seperation of LLO oligomeres by size. However the results of this thesis are good basis for further studies of LLO oligomerization and pore formation on higer level of resolution as is for example cryo-electron microscopy.

• Keywords: Listeria monocytogenes, listeriosis, proteins, toxins, citolysins, listeriolysin O, oligomers, pores, biochemical analyses