Sintezno biološki pristop za pojasnitev mehanizma delovanja in uporabo listeriolizina O

Aden, Saša (avtor), Anderluh, Gregor (mentor)

Listeriolizin O je prav poseben predstavnik družine od holesterola odvisnih citolizinov, katerega stabilnost je odvisna od pH in temperature. V bioloških sistemih je to ključna lastnost, ki pomaga bakteriji Listeria monocytogenes, da se širi po gostiteljevem organizmu. LLO je aktiven pri nizkem pH znotraj gostiteljevega fagolizosoma, kar omogoča bakteriji pobeg, medtem ko pri fiziološkem pH in temperaturi 37°C hitro agregira. Namen doktorskega dela je bilo natančneje preučiti mehanizem, s katerim LLO deluje na lipidne membrane ter te lastnosti uporabiti za aplikacije v sistemih z vezikli. Vpliv LLO na lipidne membrane smo preučevali s pomočjo modelnih membranskih sistemov, GUV-ov, z metodama pretočne citometrije in konfokalne fluorescenčne mikroskopije. Prišli smo do ugotovitev, da LLO poškoduje membrane, ki vsebujejo zadostno količino holesterola, v tolikšni meri, da vodi do propada veziklov. V biološkem sistemu to pomeni, da uničujoč učinek LLO vodi do propada fagolizosomov, kar omogoči bakteriji pobeg. Do zanimivega odkritja smo prišli tudi pri poskusih vezave LLO na arheosome, kjer se je LLO lahko vezal na arheosome, sestavljene izključno iz arhejskih lipidov. To kaže na to, da LLO poleg holesterola lahko prepoznava dodatno vezavno mesto na membranah, najverjetneje sladkor na glavah arhejski lipidov. S sintezno biološkimi pristopi smo poskusili pripraviti modelne sisteme, ki bi se specifično odzivali na spremembe v okolju z aktivacijo LLO in sproščanjem vsebine veziklov. Uspelo nam je pripraviti ogromne umetne vezikle iz arhejskih lipidov, znotraj katerih smo z IVTT uspeli proizvesti fluorescenčne fuzijske proteine z LLO oziroma Y406A. To je pomemben dosežek pri razvoju stabilnejših membran umetnih celic in korak naprej pri uporabi CDC-jev v takšnih sistemih. Sistem, ki je bil sposoben specifičnega odzivanja na spremembe v okolju, smo pripravili z uporabo proteinskih logičnih vrat na membrani veziklov. Uporabili smo od pH odvisen mutant LLO Y406A ter njegov inhibitor DARPin D22. Uspeli smo pripraviti sistema z dvema različnima logičnima funkcijama. AND logična vrata so pogojevala delovanje Y406A s prisotnostjo nizkega pH ter MMP-9. Slednja je bila potrebna za cepitev D22 is površine veziklov. OR-AND logična vrata so aktivirala tvorbo por Y406A ob cepitvi D22 is površine veziklov z reducentom TCEP ali proteazo MMP-9, in zakisanju okolice. Sintezno biološke sisteme, ki smo jih razvili, bi bilo možno uporabiti kot naprave za in vitro in in vivo dostavo, detekcijo, diagnostiko in kot ogrodje za nadaljnji razvoj umetnih celic ter membranskih sistemov z logičnimi vrati.

• Jezik: Slovenski jezik
• Ključne besede: listeriolizin O, pore, GUV, proteinska logična vrata, sintezna biologija
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2020
• PID: 20.500.12556/RUL-117536
• COBISS.SI-ID: 33941763
• Datum objave v RUL: 15.07.2020

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synthetic biology approach for elucidating mechanism of activity and use of listeriolysin O

Izvleček

Listeriolysin O is a special member among the chlesterol-dependent cytolysins, as its stability is pH and temperature dependent. This is crucial for its biological role in supporting the spread of bacteria Listeria monocytogenes in the host organism. LLO is active at low pH within the phagolysosome of the host organism, which allows bacteria to escape, and on the other hand, LLO aggregates at a phisiological pH and temperature of 37°C. The aim of the dissertation was to further elucidate the mechanism of LLO action on lipid membranes and to use it in applications including vesicle systems. We investigated the effect of LLO on lipid membranes with membrane model systems, GUV-s, by flow cytometry and fluorescent confocal microscopy. We found that LLO damages membranes containing sufficient amount of cholesterol to such an extent that it leads to destruction of vesicles. In biological systems, this means that the effect of LLO leads to the destruction of phagolysosomes, allowing the bacteria to escape. An interesting discovery was that LLO was able to bind to archaeosomes without cholesterol, which means that in addition to cholesterol, LLO has additional binding sites on the membranes, most likely sugars on the lipid heads of archaeal lipids. We used synthetic biology approaches to develop model systems that specifically activate LLO action and release vesicles cargo in response to environmental changes. We established the preparation of huge artificial vesicles composed of archaeal lipids with IVTT of fluorescent fusion proteins with LLO or Y406A inside GUV-s. This represents an important achievement in the development of stable membranes of artificial cells and a step forward in the use of CDC-s in such systems. We designed a system that cant specifically respond to environmental changes by placing protein logic gates on vesicle membranes. We used pH-dependent LLO mutanate Y406A and its inhibitor DARPin D22. We were able to design systems with two different logic functions. AND logic gates activated Y406A pore formation at low pH and in the presence of MMP-9, which cleaved D22 from the vesicle membrane. OR-AND logic gates activated Y406A pore formation upon cleavage of D22 from the vesicle membrane by the reducing agent TCEP or protease MMP-9 and the acidification of the environment. The developed synthetic biology systems could be used as devices for in vitro and in vivo delivery, detection and as a framework for the further development of artificial cells and membrane systems with logic gates.

• Ključne besede: listeriolysin O, pores, GUV, protein logic gates, synthetic biology