Necrotic enteritis is an important disease of commercial poultry, especially with increasing constraints on the prophylactic use of antimicrobial agents in animal feeds (28
). The pathogenesis of this disease is complex and remains partially unresolved (16
), but NetB is the primary toxin involved in the disease process (15
). We have now determined the crystal structure of NetB, identified residues that are essential for NetB function, and mapped these residues onto the NetB structure.
Little is known about the cells that are targeted by NetB in an intestinal infection. Initial studies tested a range of cell lines for their susceptibility to NetB, but cytopathic effects were detected only on chicken LMH cells (15
). In the current work, we have shown that NetB lyses horse RBC, and we used lytic activity on HBA to screen for netB
mutants generated by random mutagenesis. We subsequently showed that NetB was much more active on avian RBC; chicken, duck, and goose RBC all had greatly increased susceptibility to NetB-mediated lysis. The reason for this increased susceptibility is not known, it is possible that avian RBC membranes have increased levels of a cell surface receptor that is required for NetB binding and its subsequent cytolytic activity or that it may reflect differences in phospholipid composition in differing animal species.
Until recently, the structures of only two β-pore-forming toxins from C. perfringens
had been published, namely, the aerolysin-like epsilon-toxin (7
) and CPE (9
). In addition, the structure of C. perfringens
alpha-hemolysin-like delta-toxin has been deposited in the protein structure database (2YGT). Analysis of the structure of NetB indicates that it is closely related to the alpha-hemolysin family of toxins. These toxins generally form heptameric pores in the host cell membrane, and it is highly likely that NetB also forms heptameric pores on its target cell membranes.
Initial comparative sequence analysis of NetB led to the identification of D186 and R230 as residues that were likely to be of functional importance. However, site-directed mutagenesis of these residues revealed that a D186N substitution derivative was still functional. Therefore, random mutagenesis was used to identify 12 single-amino-acid substitutions that produced a stable NetB protein in C. perfringens and which were nonhemolytic on HBA. Subsequent analysis on more sensitive chicken RBC, using purified proteins, revealed that seven of these derivatives were less active than the wild-type toxin. Three of these NetB proteins, including the original R230Q derivative, were inactive against chicken RBC. The R230Q and W287R proteins were correctly folded, as determined by circular dichroism spectrometry, still formed oligomers in solution, and were able to form single channels in artificial phospholipid bilayers. Since the R230 and W287 residues are located in the putative rim domain of NetB, we postulate that these residues are required for binding to a cell surface receptor.
Unlike the other NetB substitution derivatives, the T73A, S136P, and S254L proteins all failed to form oligomers in solution. However, both the T73A and S136P proteins had reduced but significant hemolytic activity against chicken RBC, and S136P was still functional in the phospholipid bilayer assay, indicating that these residues are involved in determining the rate of spontaneous oligomerization of NetB in solution (i.e., in the absence of red cell or synthetic membranes). We suggest that these derivatives still are able to oligomerize on the surfaces of their target avian cells. In contrast, the NetB S254L derivative was unable to oligomerize and was nonfunctional in both the chicken RBC and lipid bilayer assays. In addition, comparative analysis of the NetB structure suggests that S254 is located in the NetB domain postulated to be involved in oligomerization. Therefore, we postulate that the S254 residue of NetB is essential for the formation of a functional oligomer on the surface of the target cell. The abolition of pore-forming ability by the S254L substitution is consistent with its impaired ability to oligomerize. The fact that we could detect single channels in the other four mutants tested is consistent with the substitutions lying outside the pore lumen.
The planar lipid bilayer data demonstrate that NetB has intrinsic pore-forming activity consistent with the predicted amino acid similarity to other β-pore-forming toxins. Our data also show that the electrophysiological features of NetB have both similarities to and differences from those of S. aureus
alpha-hemolysin, the PFT that has been best characterized in terms of both structure and electrophysiology. Channels formed by both NetB and alpha-hemolysin tend to remain in the open state and exhibit a tendency to close at positive voltages over +40 V. However, NetB channels are roughly 3-fold larger in conductance than those of alpha-hemolysin (27
). While alpha-hemolysin has a weak preference for anions, NetB channels exhibited a preference for cations over anions, a finding demonstrated using three experimental approaches, namely, ion replacement, reversal potential, and pH dependence. This property resembles that of a close ortholog of NetB, β-toxin from C. perfringens
, which also preferentially conducts cations over anions (33
). Our analysis of the NetB structure and that of other workers (35
) indicate that there are regions of electronegativity at both ends of the pore, which could select for cations. Significant differences in the channel conductance of NetB and alpha-hemolysin also were observed with the two phospholipids tested, phosphatidylcholine and phosphatidylserine. This result may reflect, at least in part, the amino acid sequence differences in the membrane-binding domain and the relative hemolytic potencies in the red cells from different animal species.
After this article was submitted for review, other workers (35
) reported on the molecular architecture of the NetB pore. The structure of the heptameric pore in a detergent additive, without the first 20 amino acids of mature NetB, was solved to a resolution of 3.9 Å. They did not determine the structure of soluble monomeric NetB. They also showed that NetB interacts directly with cholesterol in the target cell membrane as part of the oligomerization and pore-forming process. Since bird and mammalian RBC have similar proportions of membrane cholesterol (36
), the different susceptibilities of bird and mammalian RBC to NetB-mediated lysis observed in our study do not appear to result from major differences in cholesterol levels. Mutants equivalent to our R230Q (R200A; these authors numbered the residues based on the processed NetB protein) and W287R (W257A) derivatives were constructed and analyzed. Their results were in agreement with those of our studies, since their R200A and W257A mutants had reduced cytotoxic activity compared to that of wild-type NetB.
There is a need for new vaccines that can be used for the control and prevention of necrotic enteritis in poultry (38
). The most cost-effective necrotic enteritis vaccine may well be a live attenuated vaccine that is used in hens rather than commercial broilers. Such a vaccine may require the use of nontoxic, but immunogenic, derivatives of NetB in addition to other C. perfringens
antigens. The R230Q, S254L, and W287R substitution-containing NetB derivatives described here, which are either inactive or have very greatly reduced biological activity but are still immunoreactive, may prove to be important for the development of a protective necrotic enteritis vaccine.