Evaluation of a new cytotoxicity assay for<i>Clostridium perfringens</i>type D epsilon toxin

Payne, Dean; Williamson, E. Diane; Havard, Helen; Modi, Naren; Brown, J E

doi:10.1111/j.1574-6968.1994.tb06695.x

Cited by 64 publications

(5 citation statements)

References 17 publications

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“…ETX with different tags (GST, 6×His, and mScar) did not differ significantly in toxicity ( Figure 1 A). The ETX (GST-ETX and His-ETX) dose needed to kill ~50% of MDCK cells (CT 50 ) was 0.8 nM, and the CT 50 of mScar-ETX was 0.4 nM, in agreement with the reported CT 50 of 0.5 to 10 nM for the active wild-type ETX [ 13 , 20 , 21 , 26 , 30 ]. mScar-ETX exhibits stronger toxicity than ETX with other tags, probably because the mScar tag could enhance the stability of ETX.…”

Section: Resultssupporting

confidence: 82%

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Statins as Potential Preventative Treatment of ETX and Multiple Pore-Forming Toxin-Induced Diseases

Huang

Zhao

Liu

et al. 2023

IJMS

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Epsilon toxin (ETX), produced by type B and D strains of Clostridium perfringens, can cause fatal enterotoxaemia in ruminant animals, particularly sheep, cattle, and goats. Previous studies show that the cytotoxicity of ETX is dependent on the integrity of lipid rafts, the maintenance of which is ensured by cholesterol. Zaragozic acid (ZA) is a statin drug that reduces the synthesis of squalene, which is responsible for cholesterol synthesis. In this study, ZA significantly reduced the toxicity of ETX in Madin–Darby canine kidney (MDCK) cells. We show that ZA does not affect the binding of ETX to MDCK cells, but propidium iodide staining (PI) and Western blotting confirmed that ZA significantly disrupts the ability of ETX to form pores or oligomers in MDCK cells. Additionally, ZA decreased the phosphatidylserine exposure on the plasma membrane and increased the Ca2+ influx of the cells. Results of density gradient centrifugation suggest that ZA decreased the number of lipid rafts in MDCK membranes, which probably contributed to the attenuation of pore-formation. Moreover, ZA protected mice against ETX in vivo. All mice pre-treated with ZA for 48 h before exposure to an absolute lethal dose of ETX (6400 ng/kg) survived. In summary, these findings provide an innovative method to prevent ETX intoxication. Considering many pore-forming toxins require lipid rafts, we tested and found ZA also inhibited the toxicity of other toxins such as Clostridium perfringens Net B and β-toxin (CPB) and Staphylococcus aureus α-hemolysin (Hla). We expect ZA can thus be developed as a broad-spectrum medicine for the treatment of multiple toxins. In addition, other statins, such as lovastatin (LO), also reduced the toxicity of ETX. These findings indicate that statin medicines are potential candidates for preventing and treating multiple toxin-induced diseases.

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Section: Resultssupporting

confidence: 82%

“…Eventually, cells are killed by morphological changes, including swelling and the formation of membrane blebs [ 20 ]. The CT 50 (the dose needed to kill 50% of cells) of ETX in MDCK cells is 15 ng/mL [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

Statins as Potential Preventative Treatment of ETX and Multiple Pore-Forming Toxin-Induced Diseases

Huang

Zhao

Liu

et al. 2023

IJMS

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“…discovered that a cell culture assay employing MDCK cells, purified Etx diluted in culture medium and specific neutralizing antibodies (monoclonal) against Etx correlates well with the mouse lethal assay [77]. Of the twelve different cell lines tested, the MDCK is the only one susceptible (~15 ng Etx/mL detection limit).…”

Section: A Veterinary Perspective On Etx: Field and Laboratory Finmentioning

confidence: 99%

Clostridium perfringens Epsilon Toxin: A Malevolent Molecule for Animals and Man?

Stiles

Barth

et al. 2013

Toxins

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Clostridium perfringens is a prolific, toxin-producing anaerobe causing multiple diseases in humans and animals. One of these toxins is epsilon, a 33 kDa protein produced by Clostridium perfringens (types B and D) that induces fatal enteric disease of goats, sheep and cattle. Epsilon toxin (Etx) belongs to the aerolysin-like toxin family. It contains three distinct domains, is proteolytically-activated and forms oligomeric pores on cell surfaces via a lipid raft-associated protein(s). Vaccination controls Etx-induced disease in the field. However, therapeutic measures are currently lacking. This review initially introduces C. perfringens toxins, subsequently focusing upon the Etx and its biochemistry, disease characteristics in various animals that include laboratory models (in vitro and in vivo), and finally control mechanisms (vaccines and therapeutics).

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“…The efficacy of the antisera generated against the rEtx was determined by in vitro neutralization assays using MDCK cells which are susceptible to Etx toxicity (Payne et al 1994). Notable reduction in the rEtx toxicity was noted when it was pre-incubated with different dilutions of anti-fusion protein antisera prior to its addition to the cells ( p ≤ 0.005 to p ≤ 0.001).…”

Section: Resultsmentioning

confidence: 99%

Immunization with recombinant fusion of LTB and linear epitope (40–62) of epsilon toxin elicits protective immune response against the epsilon toxin of Clostridium perfringens type D

et al. 2019

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Epsilon toxin (Etx) produced by Clostridium perfringens types B and D, a major causative agent of enterotoxaemia causes significant economic losses to animal industry. Conventional vaccines against these pathogens generally employ formalin-inactivated culture supernatants. However, immunization with the culture supernatant and full length toxin subjects the animal to antigenic load and often have adverse effect due to incomplete inactivation of the toxins. In the present study, an epitope-based vaccine against Clostridium perfringens Etx, comprising 40–62 amino acid residues of the toxin in translational fusion with heat labile enterotoxin B subunit (LTB) of E. coli , was evaluated for its protective potential. The ability of the fusion protein rLTB.Etx 40–62 to form pentamers and biologically active holotoxin with LTA of E. coli indicated that the LTB present in the fusion protein retained its biological activity. Antigenicity of both the components in the fusion protein was retained as anti-fusion protein antisera detected both the wild type Etx and LTB in Western blot analysis. Immunization of BALB/c mice with the fusion protein resulted in a significant increase in all isotypes, predominantly IgG1, IgG2a and IgG2b. Anti-fusion protein antisera neutralized the cytotoxicity of epsilon toxin both in vitro and in vivo. Thus, the results demonstrate the potential of rLTB.Etx 40–62 as a candidate vaccine against C. perfringens . Electronic supplementary material The online version of this article (10.1186/s13568-019-0824-3) contains supplementary material, which is available to authorized users.

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Evaluation of a new cytotoxicity assay forClostridium perfringenstype D epsilon toxin

Cited by 64 publications

References 17 publications

Statins as Potential Preventative Treatment of ETX and Multiple Pore-Forming Toxin-Induced Diseases

Statins as Potential Preventative Treatment of ETX and Multiple Pore-Forming Toxin-Induced Diseases

Clostridium perfringens Epsilon Toxin: A Malevolent Molecule for Animals and Man?

Immunization with recombinant fusion of LTB and linear epitope (40–62) of epsilon toxin elicits protective immune response against the epsilon toxin of Clostridium perfringens type D

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