Listeriolysin O Membrane Damaging Activity Involves Arc Formation and Lineaction -- Implication for Listeria monocytogenes Escape from Phagocytic Vacuole.
Listeriolysin-O (LLO) plays a crucial role during infection by Listeria monocytogenes. It enables escape of bacteria from phagocytic vacuole, which is the basis for its spread to other cells and tissues. It is not clear how LLO acts at phagosomal membranes to allow bacterial escape. The mechanism of action of LLO remains poorly understood, probably due to unavailability of suitable experimental tools that could monitor LLO membrane disruptive activity in real time. Here, we used high-speed atomic force microscopy (HS-AFM) featuring high spatio-temporal resolution on model membranes and optical microscopy on giant unilamellar vesicles (GUVs) to investigate LLO activity. We analyze the assembly kinetics of toxin oligomers, the prepore-to-pore transition dynamics and the membrane disruption in real time. We reveal that LLO toxin efficiency and mode of action as a membrane-disrupting agent varies strongly depending on the membrane cholesterol concentration and the environmental pH. We discovered that LLO is able to form arc pores as well as damage lipid membranes as a lineactant, and this leads to large-scale membrane defects. These results altogether provide a mechanistic basis of how large-scale membrane disruption leads to release of Listeria from the phagocytic vacuole in the cellular context.
U1006 INSERM, Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, Marseille, France.
Yi
Y
Ruan
Department for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Saša
S
Rezelj
Department for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Apolonija
A
Bedina Zavec
Department for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia.
Gregor
G
Anderluh
U1006 INSERM, Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, Marseille, France.
Simon
S
Scheuring
D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D005434:Flow Cytometry; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D008089:Listeria monocytogenes / Q000472:pathogenicity*; D008088:Listeriosis / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D010588:Phagosomes / Q000382:microbiology*
D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D002784:Cholesterol; C038705:hlyA protein, Listeria monocytogenes