Date Published: April 22, 2016
Publisher: Public Library of Science
Author(s): Yi Ruan, Saša Rezelj, Apolonija Bedina Zavec, Gregor Anderluh, Simon Scheuring, Mary O’Riordan.
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.
Listeriolysin-O (LLO) is Listeria monocytogenes powerful molecular weapon in host cell invasion, which is the first step of the disease listeriosis . Following accidental ingestion of Listeria-contaminated food, healthy humans suffer from gastroenteritis, while immunocompromised individuals are affected in the nervous system and can suffer severe damage. Listeria infection is treated by antibiotics, but as the development of novel antibiotics is a serious bottleneck, an improved understanding of LLO action may provide novel angles of attack to fight against this disease.
Controlled HS-AFM movie acquisition at sub-second temporal resolution of LLO assembly on model membranes allowed analyzing its action in a series of well-defined experimental conditions, notably when the protein was exposed to bilayers containing varying amounts of cholesterol (along with DOPC) and as a function of environmental pH.
Here we present a first dynamic analysis of LLO membrane activity at high-spatio-temporal resolution. A DOPC/Cholesterol model membrane system was used at various cholesterol content bathing in buffers of various pH. These experimental conditions combined with the capacity of dynamic imaging, allowed us to acquire a detailed understanding of the molecular action of LLO.