NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

Abstract

For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB) was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE). The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity) and alpha-toxin from Staphylococcus aureus (31% identity). NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

Figure 1. Cytotoxic Activity of EHE-NE18 Supernatant on LMH Cells

The LMH cells were grown to 70% confluence and culture supernatant added to the medium as a 2-fold dilution series across the plate and incubated for up to 16 h at 37 °C. Cytopathic effects were observed under a light microscope at 100× magnification. (A) TPG culture medium (undiluted); (B) EHE-NE18 culture supernatant (1:16 dilution); (C) JIR325 culture supernatant (1:2 dilution); (D) NE18-M1 (plc mutant) culture supernatant (1:16 dilution). (B) And (D) show obvious cell rounding and detachment.

Figure 2. SDS-PAGE and Western Blot of Purified Native NetB and rNetB Toxin

Proteins were separated by 4%–12% SDS-PAGE. Native NetB was prepared with Sepharose Q FF anion exchange resin and rNetB-toxin was purified on a nickel affinity column followed by gel filtration. SeeBlue Plus2 prestained marker (Invitrogen) was used as a size marker. (A) SDS-PAGE stained with coomassie blue: M, SeeBlue Plus2 marker; NetB, native NetB; rNetB, recombinant NetB. (B) SDS-PAGE transferred onto PVDF membrane and probed with rabbit polyclonal anti-rNetB antibody. Blots were developed with an ECL western blotting kit and the results recorded on autoradiographic film: NetB, native NetB; rNetB, recombinant NetB.

Figure 3. ClustalW Alignment of NetB

ClustalW alignment of the toxins C. perfringens NetB (EU143239), C. perfringens beta-toxin (Cpb, AAA23284.1), B. cereus hemolysin II (Hly-II, NP_833256.1), and S. aureus alpha-toxin (Hla, NP_371687.1). Identical residues (*), conservative amino acid substitutions (:), and semi-conservative amino acid substitutions (.) are shown below the aligned sequences. To compact the figure the Hly-II sequence was shortened (//).

Figure 4. Phylogenetic Analysis of NetB

Phylogenetic analysis of representative members of the “S. aureus pore-forming toxin family” with beta2-toxin (Cpb2) used as outliers. Clustal X with neighbour-joining bootstrapping (1000 interactions) was used to construct the tree. Toxins that were used included: Beta-toxin (Cpb) from C. perfringens (CAA58246.1, AAA23284.1, CAB75343.1); Hla from S. aureus (NP_371687.1, YP_186036.1, 7AHLA, P09616, NP_645861.1); HlyII from B. cereus (AAB51536.1, YP_083859.1, NP_833256.1, AAM21564.1); B. thuringiensis (YP_895080.1, YP_036637.1); B. anthracis (YP_028614.1); CytK from B. cereus (ABI52591.1, YP-001374171.1); B. thuringiensis (ZP_00739479.1); and LukF from S. aureus (P31715); S. intermedius (CAA55783.1). Beta2-toxin (Cpb2) from C. perfringens (CAD61052.1, CAD61053.1, CAD61054.1, AAW79185.1, AAW80352.1, AAW80353.1, AAW80354.1, AAW80355.1, AAW80356.1, AAW80357.1, AAW80358.1).

Figure 5. Cytotoxic Activity of Supernatants from EHE-NE18, EHE-NE18ΔnetB1, and Complemented Mutants

The LMH cells were cultured until 70% confluence in 24 well plates coated in 0.2% gelatine and grown in EMEM medium at 37 °C. (A) Cytotoxicity assays. Culture supernatant was added to the medium and incubated for up to 16 h at 37 °C. (1) EHE-NE18 (1:16 dilution); (2) NE18ΔnetB1 (1:2 dilution); (3) NE18ΔnetB1(pJIR1457) (shuttle plasmid)(1:2 dilution); (4) NE18ΔnetB1(pALK20) (netB⁺ complementation plasmid) (1:16 dilution); (5) TPG culture medium (undiluted); (6) Purified rNetB (0.5 μg). (B) Neutralisation of NetB cytotoxicity. Pre-immune sera and antisera raised against rNetB were incubated with 0.5 μg/ml of purified native NetB-toxin (1:20) for 1 h at room temperature. Treated and non-treated samples where added to LMH cells and incubated for 16 h at 37 °C. Cytopathic effects were observed under the light microscope at 100× magnification. (1) NetB positive control; (2) NetB pretreated with pre-immune rabbit sera; (3) NetB pretreated with rabbit anti-rNetB antiserum.

Figure 6. Lactate Dehydrogenase Cytotoxicity Assay of LMH Cells Treated with NetB and NetB Pore-size Estimation

(A) Purified NetB was added to the tissue culture medium with two-fold dilutions up to 62.5 ng of toxin and incubated with LMH cells for 4 h at 37 °C. The percentage of total LDH released in the supernatant was measured with a Cyto-Tox (Promega) kit and used as an indicator of cytolysis. Each dilution was assayed in triplicate in three independent experiments. (B) LMH cells were incubated with 0.5 μg/well protein and 25 mM PEGs with different molecular sizes for 4 h, and the extent of cellular lysis was determined by measuring the amount of LDH released. The results are presented as a percentage of the control (cells without any PEG). The values are averages of triplicate assays in three experiments with error bars representing SEM.

Figure 7. Southern Hybridization Analysis of HindIII-digested Genomic DNA

DNA from EHE-NE18 (1) and NE18ΔnetB1 (2) was probed with DIG-labelled DNA specific for the netB (A) and catP (B) genes as shown. Appropriate lambda DNA size markers are arrowed and labelled at the left.

Figure 8. Virulence of C. perfringens Strains in the NE Challenge Model

The lesion scores of individual 24-day-old broiler chickens challenged with different C. perfringens strains are shown. Each group consisted of ten birds. The solid horizontal bars represent the average lesion score in each group. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously reported [9]: 0, no gross lesions; 1, thin or friable walls; 2, focal necrosis or ulceration (one to five foci); 3, focal necrosis or ulceration (six to 15 foci); 4, focal necrosis or ulceration (16 or more foci); 5, patches of necrosis 2–3 cm long; 6, diffuse necrosis typical of field cases. The results are from two separate trials. The strains tested are as follows: EHE-NE18, wild-type; ΔnetB, NE18ΔnetB1; ΔnetB Comp, NE18ΔnetB1(pALK20); ΔnetB1 + pJIR1457, NE18ΔnetB1(pJIR1457). One-tailed, nonparametric t-test analysis of the challenge (EHE-NE18) and complemented mutant derivatives against NE18ΔnetB1 showed a statistical difference (**, p < 0.01 and *, p < 0.05) but no statistical significance was observed between the mutant and the plasmid control.