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.

Structures of Toxoplasma perforin-like protein facilitate understanding of the protein’s role in infection and disease.

Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.

Archaeosomes are vesicles made of lipids from archaea. They possess many unique features in comparison to other lipid systems, with their high stability being the most prominent one, making them a promising system for biotechnological applications. Here, we report a preparation protocol of large unilamellar vesicles, giant unilamellar vesicles (GUVs), and nanodiscs from archaeal lipids with incorporated cholesterol. Incorporation of cholesterol led to additional increase in thermal stability of vesicles. Surface plasmon resonance, sedimentation assays, intrinsic tryptophan fluorescence measurements, calcein release experiments, and GUVs experiments showed that members of cholesterol-dependent cytolysins, listeriolysin O (LLO), and perfringolysin O (PFO), bind to cholesterol-rich archaeosomes and thereby retain their pore-forming activity. Interestingly, we observed specific binding of LLO, but not PFO, to archaeosomes even in the absence of cholesterol. This suggests a new capacity of LLO to bind to carbohydrate headgroups of archaeal lipids. Furthermore, we were able to express LLO inside GUVs by cell-free expression. GUVs made from archaeal lipids were highly stable, which could be beneficial for synthetic biology applications. In summary, our results describe novel model membrane systems for studying membrane interactions of proteins and their potential use in biotechnology.