Study of membrane binding and membrane insertion mechanisms of Listeriolysin O a prominent member in the cholesterol dependent cytolysin family of barrel pore forming toxins

Abstract

Listeriolysin O (LLO) is a prominent member in the family of cholesterol-dependent newlinecytolysins (CDCs), and a crucial virulence factor secreted by Listeria monocytogenes. The pore- newlineforming activity of LLO is vital for the pathogenesis of L. monocytogenes. The pore-formation newlinemechanism of LLO, like that of other CDCs, is dependent on membrane cholesterol. It involves a newlineseries of complex conformational reorganizations, making the mechanism challenging to fully newlinecomprehend. The present study has explored the mechanistic basis of LLO binding to the membrane, newlineand the membrane-insertion of its pore-forming motif to form the functional pores. In the first part of newlinethe study, we have investigated the roles of cholesterol in facilitating LLO activity and examined the newlinemechanistic basis of the LLO-cholesterol interaction. Here, we show that cholesterol promotes both newlinemembrane binding and oligomerization of LLO. Furthermore, the binding of LLO is dependent on the newlinemembrane composition and dynamics, particularly in cholesterol-deficient membranes. To understand newlinethe mechanistic basis of LLO-cholesterol interactions, we employed an LLO variant in which the newlinecholesterol-recognition motif was altered (LLO T515G-L516G ). Interestingly, we find that the membrane- newlinebinding and pore-forming abilities of LLO T515G-L516G , but not those of LLO, correlate with the newlinecholesterol-dependent ordering of the lipid bilayer. Our data further suggest that the line tension newlinearising from the lipid phase heterogeneity of cholesterol-containing membranes could play a pivotal newlinerole in LLO function, particularly in the absence of CRM-mediated cholesterol binding. Therefore, in newlineaddition to its receptor-like role, we conclude that cholesterol further facilitates the pore-forming and newlinemembrane-damaging functionality of LLO by establishing the optimal physicochemical environment newlinein the membranes. In the second part of this study, we have explored the structural and functional newlinesignificance of the two transmembrane helices (TMHs), TMH1 and TMH2, in LLO pore-formation. newlineW