Cholesterol
dependent citolysins (CDCs) are a family
of pore-forming proteins, expressed by various bacterial species. They are all
secreted as soluble monomers that bind to membrane cholesterol, oligomerise and
form pores up to 35 nm in diameter. Pore formation triggers also numerous other
cellular effects, such us endocytosis, histone modification, activation of
kinases and others. Lately our study have been focused on listeriolysin O, a pH
dependent CDC that is expressed by human pathogen G+ bacteria Listeria monocytogenes. The bacteria
causes listeriosis, which starts with the ingestion of contaminated foods and
affects mainly immunocompromised persons and pregnant women. Infection during
pregnancy can result in life-threatening infection of the newborns, and also 30
% of infections in adults result in death. Besides being the main viruence
factor of Listeria, is LLO interesting
also for its pH dependent pore-forming activity which has a potential to be used
for targeted drug delivery system in a combination with suitable membrane
sistem therefore the understanding of its membrane binding events is crucial
for further experiment design. For LLO's membrane binding study several artificial
membrane systems with high cholesterol level were prepared (large, small and
giant multilamellar vesicles, and nanodiscs) with different lipid composition. Calcein
release, centrifugation assay, hemolysis, SPR and NMR experiments were
performed to gain more insight into binding at molecular level. LLO was also
labelled with Alexa Fluor A488 and its binding to artificial membrane vesicles
was observed with confocal microscopy. We confirmed LLO's specific cholesterol-dependent
binding and introduced new techniques in lipid vesicles preparation, such as
nanodiscs, composed of archaeal lipids w/o cholesterol, which are of special
interest since arcaeal lipids are known to be among most stable one.