Effect of zinc and calcium ions on the production of alpha-toxin and proteases by Clostridium perfringens

Abstract

Clostridiumperfringens produced at least three distinct proteases in a synthetic medium containing calcium. Two of them, thiol and ethylenediaminetetraacetic acid disodium salt-sensitive proteases, appeared at an early stage of growth, but the other one, perhaps being identical to the one produced in a calcium-deficient medium, appeared at a late stage. The production of these proteases depended on Ca2" but not on Zn2+ in the medium. Alpha-toxin, perhaps being a zinc-containing metalloenzyme, was rather resistant to the proteases, but toxin, produced in a zinc-deficient medium or deprived of zinc with ethylenediaminetetraacetic acid disodium salt, was very sensitive. By adding Zn2+, the toxin lacking zinc may have been converted to the zinc-containing metalloprotein that is resistant to proteases. This may explain why a-toxin activity increased progressively in a zinc-containing medium in spite of simultaneous production of potent proteases and why it disappeared rapidly in a zinc-deficient medium.

The carboxy‐terminal C2‐like domain of the α‐toxin from Clostridium perfringens mediates calcium‐dependent membrane recognition

“…No evidence for such a site was obtained by equilibrium dialysis, gel electrophoresis under nondenaturing conditions or column chromatography (data not shown), suggesting that, if a calcium-binding site exists, it must be of extremely low affinity. This is entirely consistent with the fact that very high calcium concentrations are required for optimal enzyme activity with physiological substrates (Sato et al, 1978).…”

“…Progress curves obtained under different calcium concentrations using purified recombinant protein at 0.5 g ml ¹1 are shown. In the absence of calcium, no change in turbidity was observed, consistent with the obligate requirement of ␣-toxin for this cation (Sato et al, 1978). Use of calcium concentrations > 5.2 mM led to no significant increase in activity.…”

“…The phospholipase C activity of C. perfringens ␣-toxin is absolutely dependent on calcium (Sato et al, 1978;Fig. 2).…”

“…Notably, replacement of the catalytic aspartate residue at position 56 by asparagine resulted in the abolition of all measurable biological activities (haemoloysis, phosphatidylcholine and sphingomyelin hydrolysis, platelet aggregation) and concomitant detoxification. In addition to zinc, ␣-toxin also requires calcium ions for full activity (Sato et al, 1978) although there is a striking difference in the optimal concentrations (Zn 2þ , 5 M; Ca 2þ , Ϸ5 mM). In a seminal study, Moreau et al (1988) demonstrated clearly that calcium ions are essential for the binding of ␣-toxin to lipid films, whereas zinc is required for substrate hydrolysis.…”

Bacterial phospholipases C.

“…Zinc-Metalloenzymes The B. cereus PC-PLC, C. perfringens alpha-toxin, Clostridium bifernentans PLC, Listeria monocytogenes PLC-B, and Clostridium novyi gamma-toxin form a group of related enzymes which contain essential zinc ions and are reversibly inactivated by EDTA or o-phenanthroline (54,64,76,143,160,169). It also seems likely that the phospholipases C produced from Clostridium absonum and Clostridium barati, which are antigenically and genetically related to the C. perfringens alpha-toxin (112,171), are also zinc-metallophospholipases C.…”