Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease

Abstract

Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases.

Figure 1. Crystal structures of Clostridium perfringens toxins

Graphic representations of all known toxin structures were prepared using Pymol [88]. Color coding is shown as a transition from N-terminal (blue) to C-terminal [35] residues. Note that delta toxin is a pore-forming toxin, but its role in disease has not yet been established. CPA: C. perfringens alpha toxin (1CA1) [89]; CPE: C. perfringens enterotoxin (3AM2) [40,41]; Delta: Delta toxin (2YGT) [93]; ETX: C. perfringens epsilon toxin (1UYJ) [92]; ITXa: C. perfringens iota toxin a subunit (1GIR) [90]; NetB: Necrotic enteritis toxin B (4ION) [50]; PFO: Perfringolysin O (1PFO) [91]. For color images please see www.futuremedicine.com/doi/full/10.2217/fmb.13.168

Figure 2. Microscopic lesions in mice inoculated with Clostridium perfringens type A (mouse gas gangrene model)

The inner tight muscle shows severe necrosis of myofibers characterized by flocculation and fragmentation of the sarcolemma, hypereosinophilia and nuclear pyknosis. A mid-size venule (*) shows marked margination of neutrophils, which are mostly absent in the extravascular tissue. Sections were stained with hematoxylin and eosin and photographed at 100× magnification. Modified with permission from [26].

Figure 3. Histological damage in rabbit ileum treated with lysates from Clostridium perfringens enterotoxin-positive C. perfringens type A strain SM101

Loops inoculated with WT SM101 vegetative culture (WT vegetative) or an isogenic CPE KO sporulating culture lysate (CPE KO sporulating) show normal, full-length intestinal villi with a well-preserved epithelium and lamina propria. Loops inoculated with sporulating culture lysate of WT SM101 (WT sporulating) or the isogenic CPE complement (CPE complement sporulating) show histological damage consisting of necrosis and loss of epithelium, necrosis of lamina propria, villous blunting, and hemorrhage and edema of the mucosa and submucosa. Sections were stained with hematoxylin and eosin and photographed at 250× magnification. CPE: C. perfringens enterotoxin; KO: Knockout; WT: Wild-type. Figure and legend modified with permission from [44].

Figure 4. Virulence of Clostridium perfringens type A strains in 24-day-old broiler chickens challenged with different C. perfringens strains are shown

Solid horizontal bars depict the mean small intestinal lesion score in each group (n = 10). Lesion scores correspond to 0: no gross lesions; 1: thin and/or friable walls; 2: focal necrosis or ulceration (1–5 foci); 3: focal necrosis or ulceration (6 to 15 foci); 4: focal necrosis or ulceration (>16); 5: patches of necrosis 2–3 cm long; 6: diffuse necrosis typical of field cases. The strains tested included: WT; netB KO, netB KO complemented (netB KO complement); netB knockout + shuttle vector (netB KO + shuttle vector). One tailed, nonparametric t-test analysis of the wild-type and complemented mutant derivatives against netB KO showed a statistical difference (*p = 0.05 and **p = 0.01), but no statistical significance was observed between the netB KO and the netB KO + shuttle vector. KO: Knockout; WT: Wild-type. Figure and legend modified from [49].

Figure 5. Gross pathology of rabbit-ligated small intestinal loops challenged for 6 h with Clostridium perfringens type C strain CN3685 WT, isogenic single- and double-toxin mutants (PFO KO, CPA KO, CPB KO, CPA/PFO KO), purified CPB (CPB) or sterile tryptic soy broth–glucose–yeast extract broth (control)

Note that loops inoculated with the WT, PFO KO, CPA KO, CPA/PFO KO or purified CPB are severely hemorrhagic and distended with fluid. No significant gross abnormalities are observed in the loops inoculated with CPB KO or sterile tryptic soy broth–glucose–yeast extract broth. CPA: C. perfringens alpha toxin; CPB: C. perfringens beta toxin; KO: Knockout; PFO: Perfringolysin O; WT: Wild-type. Modified with permission from [76].

Figure 6. Histological damage in rabbit small intestine treated for 6 h with an 8-h culture of WT strain CN3685 (WT), single- and double-toxin isogenic mutants (PFO KO, CPA KO, CPB KO, CPA/PFO KO), or sterile tryptic soy broth–glucose–yeast extract broth medium (control)

Control loops and loops inoculated with the CPB KO showed normal, full-length intestinal villi with a well-preserved epithelium and lamina propria. Loops inoculated with WT, CPB reversed mutant, PFO KO, CPA KO, or CPA/PFO KO all showed microscopic changes, including mucosal necrosis, hemorrhage and blunting of the villi, together with neutrophilic infiltration of mucosa and submucosa. Tissue sections were stained with hematoxylin and eosin, and photographed at 200× magnification. CPA: C. perfringens alpha; CPB: C. perfringens beta; KO: Knockout; PFO: Perfringolysin O; WT: Wild-type. Figure and legend modified with permission from [76].

Figure 7. Kaplan–Meier survival curves for sheep, goats and mice Clostridium perfringens type D infection models

Survival over 24 h in (A) sheep and (B) goats or 48 h (C) in mice after intraduodenal treatment with the WT strain CN1020 (WT), the etx mutant JIR4981 (ETX knockout), its complemented derivative JIR12604 (ETX complemented), or TGY. Each inoculum was administered to six sheep, five goats and 15 mice. etx: C. perfringens epsilon; TGY: Tryptic soy broth–glucose–yeast extract broth; WT: Wild-type. Figure and portions of the legend used with permission from [83].

Figure 8. Microscopic lesions in sheep after intraduodenal treatment with Clostridium perfringens type D WT strain CN1020 (WT) or isogenic derivatives including an etx null mutant (etx KO), its complemented derivative (etx complement), or sterile, nontoxic culture medium (control) are shown

Brains from sheep challenged intraduodenally with the WT or complemented strains show proteinaceous perivascular edema (arrow) due to increased vascular permeability. Significant histological lesions were absent from the brains of sheep treated intraduodenally with the etx KO or control animals. etx: C. perfringens epsilon; KO: Knockout; WT: Wild-type. Figures and portions of the legend reproduced with permission from [83].

Figure 9. Summary of toxin involvement in Clostridium perfringens disease

(A) During histotoxic infections CPA plays a predominant role after C. perfringens (blue rods) are introduced by trauma into muscle tissue. This toxin induces localized necrosis, as well as toxemic effects in other organs when toxins enter the circulation. Both CPA and PFO are important in vascular leukostasis. (B) During intestinal infections, C. perfringens (blue rods) can produce several pore-forming toxins, including CPE during sporulation or (during vegetative growth) NetB, ETX or CPB. Host intestinal protease levels can either destroy (e.g., CPB) or activate (e.g., ETX) these toxins. When present in an active form, these toxins can cause local damage (necrosis) or be absorbed into the circulation to damage internal organs such as the brain, kidney or liver. CPA: C. perfringens alpha toxin; CPB: C. perfringens beta toxin; CPE: C. perfringens enterotoxin; PFO: Perfringolysin O. For color images please see www.futuremedicine.com/doi/full/10.2217/fmb.13.168