Cholesterol stimulates the lytic activity of Adenylate Cyclase Toxin on lipid membranes by promoting toxin oligomerization and formation of pores with a greater effective size

Several toxins acting on animal cells present different, but specific, interactions with cholesterol. Bordetella pertussis infects the human respiratory tract and causes whooping cough, a highly contagious and resurgent disease. Its virulence factor adenylate cyclase toxin (ACT) plays an important role in the course of infection. ACT is a pore‐forming cytolysin belonging to the Repeats in ToXin (RTX) family of leukotoxins/hemolysins and is capable of permeabilizing several cell types and lipid vesicles. Previously, we observed that in the presence of cholesterol ACT induces greater liposome permeabilization. Similarly, recent reports also implicate cholesterol in the cytotoxicity of an increasing number of pore‐forming RTX toxins. However, the mechanistic details by which this sterol promotes the lytic activity of ACT or of these other RTX toxins remain largely unexplored and poorly understood. Here, we have applied a combination of biophysical techniques to dissect the role of cholesterol in pore formation by ACT. Our results indicate that cholesterol enhances the lytic potency of ACT by promoting toxin oligomerization, a step which is indispensable for ACT to accomplish membrane permeabilization and cell lysis. Since our experimental design eliminates the possibility that this cholesterol effect derives from toxin accumulation due to lateral lipid phase segregation, we hypothesize that cholesterol facilitates lytic pore formation, by favoring a toxin conformation more prone to protein–protein interactions and oligomerization. Our data shed light on the complex relationship between lipid membranes and protein toxins acting on these membranes. Coupling cholesterol binding, increased oligomerization and increased lytic activity is likely pertinent for other RTX cytolysins. Here, we show that cholesterol enhances the lytic potency of the adenylate cyclase toxin (ACT) by promoting toxin oligomerization, an indispensable step for ACT to accomplish membrane permeabilization and cell lysis. Our data further show that the pores formed by ACT in cholesterol containing membranes are proteolipidic pores of greater effective size. Coupling cholesterol binding, increased oligomerization and increased lytic activity is likely pertinent for other RTX cytolysins.