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

Abstract

Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily 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 factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus 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 recognize 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 differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin.

Toxin Plasmids of Clostridium perfringens

“…Although the precise NetB receptor has not been identified, there is evidence for cell specificity, since not all chicken cell lines are susceptible to NetB (11). Recent studies have shown that NetB interacts with cholesterol to enhance pore formation (134) and that it formed pores with much higher single-channel conductance than alpha-hemolysin and varied in its ion selectivity, preferring cations over anions (133).…”

Progress and problems in vaccination against necrotic enteritis in broiler chickens

“…After the structure and function of the NetB toxin protein were analysed, mutants with reduced cytotoxic activity were designed Yan et al, 2013). The mutation of tryptophan to alanine at position 262 (W262A) resulted in a significant reduction in cytotoxicity to LMH cells and haemolytic activity on red blood cells, and thus suggested it as a vaccine candidate .…”

Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity and In Vitro Cytotoxicity Activity

“…Using this threading technique, 3 of the 4 amino acid substitutions can be modeled; CPB is slightly larger than NetB (309 versus 293 amino acids, respectively) so the K40N substitution could not be modeled, as it is absent from NetB. This model predicts that the A300V switch occurs on one of 4 loops located near the bottom of the toxin, correlating with the predicted binding or rim domain of NetB (26,30). It appears likely that, based on the homology with NetB and our result, these 4 loops create the binding domain of CPB, although more work is needed to confirm this hypothesis.…”

“…Since the alteration of a single amino acid can affect the function of clostridial pore-forming toxins (21,23,(26)(27)(28), our current work identified natural CPB sequence variations and assessed their effects on the stability of these CPB variants in the presence of trypsin and on their CPB cytotoxicity in vitro.…”