Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens

X.-X.Y. and C.J.P. contributed equally to the research and are joint first authors. J.C.W. and J.I.R. contributed equally to the research and are joint senior authors. ABSTRACT Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, pri-marily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence fac-tor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a -barrel pore-forming toxin from Staphy-lococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may rec-ognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB dif