Essential role of domain 4 of pneumolysin from Streptococcus pneumoniae incytolytic activity as determined by truncated proteins

Pneumolysin (PLY), an important virulence factor of Streptococcus pneumonia e, is one of the members of thiol-activated cytolysins (TACYs) consisting o f four domains. TACYs commonly bind to membrane cholesterol and oligomerize to form transmembrane pore. We have constructed full-length and various tr uncated PLYs to study the role of domains of PLY in the cytolytic activity. Full-length PLY had binding ability to both cell membrane and immobilized cholesterol. A truncated PLY which comprised only domain 4 molecule, the C- terminal domain of PLY, sustained the binding ability to cell membrane and cholesterol, whereas domain 1-3 molecule had no binding ability to them. Fu rthermore, the domain 4 molecule inhibited both the membrane binding and th e hemolytic activity of full-length PLY. Accordingly, the present results p rovided the direct evidence that domain 4 was essential for the initial bin ding to membrane cholesterol and the interaction led to the subsequent memb rane damage process. (C) 2001 Academic Press.