Garcia-Linares, S. | Search bioRxiv

bioRxiv2025

Recombinant production in Escherichia coli of functionally active alpha-hemolysin from the human pathogen Staphylococcus aureus

Staphylococcus aureus is a human opportunistic pathogen capable of causing multiple infections in both humans and animals. It secretes a group of exotoxins, known as hemolysins, which are released to enhance its pathogenicity. All of them exhibit cytolytic activity on a variety of host cell types, but -hemolysin stands out for being the most thoroughly studied variant. In this work, we show the production and purification of S. aureus -hemolysin following a straightforward protocol and in sufficient quantity to consider it as a potential procedure for future biotechnological approaches. Functional and structural characterization has indeed revealed that the protein is fully functional, confirming the key role of cholesterol in the necessary protein-lipid interaction. Furthermore, it has also been shown that the purified toxin can be assembled into single-particle individual pores within soluble lipid platforms in the form of cholesterol-containing nanodiscs.

biochemistry↗

bioRxiv2024

The action mechanism of actinoporins revealed through the structure of pore-forming intermediates

Pore-forming proteins exemplify the transformative potential of biological molecules. Initially produced in a monomeric, water-soluble form, they spontaneously assemble into multimeric integral membrane proteins in the presence of suitable target lipids. Their functions include roles in apoptosis, cell signaling, immunity, as well as attack and defense systems between different organisms. This latter group encompasses actinoporins, a family of pore-forming toxins from sea anemones that kill target cells by perforating their plasma membrane. Here, we have determined the structures of two such toxins, fragaceatoxin C and sticholysin II, in a membrane environment using cryogenic electron microscopy. The structures reveal how dozens of lipid molecules interact in an orderly manner, forming an intrinsic part of the pore. We have also isolated different pore-forming intermediates, where only a fraction of the constituent monomers is incorporated, exhibiting non-closed, arc-shaped structures. Based on these structures we propose a mechanism of action where the sequential assembly of toxin monomers onto the membrane, accompanied by conformational changes, triggers pore formation and membrane perforation. Our results contribute to a better understanding of the transforming capacity of these pore-forming proteins, which are becoming increasingly important for their diverse biotechnological applications.

biochemistry↗