Purification and partial characterization of the exotoxin of Corynebacterium ovis

Abstract

1. The toxin from Corynebacterium ovis, a phospholipase D (sphingomyelin phosphodiesterase D) that acts on 2-lysophosphatidylcholine and sphingomyelins, was purified by about 400-fold to homogeneity as judged by several criteria. [The EC number of the toxin (EC 3.1.4.41) has been allotted by the Nomenclature Committee of IUB, but has not yet been published.] 2. A new assay method performed in vitro, based on inhibition by the toxin of erythrocyte lysis by staphylococcal beta-haemolysin, was developed to facilitate the purification. 3. The toxin was found to be a basic (pI9.1) glycoprotein of mol.wt. 14,500 +/- 1,000. 4. The amino acid composition of the toxin was highly reminiscent of that of collagen, since it contained hydroxyproline, hydroxylysine and a high proportion of glycine, but preliminary tests showed no other similarities to collagen or proteins with similar compositions.

The exotoxins ofCorynebacterium ulcerans

“…It is possible that the purified toxin was in fact C. ovis toxin. The estimated molecular weight (13 000-15000) corresponds fairly closely with that of C. ovis toxin (14000 + 1000 (Onon, 1979)). The pathological changes produced in guinea-pigs by subcutaneous inoculation of lethal doses of C. ovis and diphtheria toxins are not readily distinguished when very high doses are employed, but with moderate doses which cause death after 48 h or longer, diphtheria toxin produces characteristic congestion and haemorrhage in the adrenal cortex.…”

“…Although C. ovis grows readily in many types of broth in common use (including those employed for production of diphtheria toxin), special media are required for good yields of toxin. We have used mainly 'Medium I', a brain-heart infusion with added lactalbumin hydrolysate and yeast extract (details in Onon, 1979). Best yields are obtained by providing liberal aeration either by shaking or by vortex aeration with a magnetic stirrer during incubation at 37 'C.…”

“…Titration of C. ovis toxin by inhibition of staphylococcal beta-haemolysin Inhibition ofthe action ofstaphylococcal beta-haemolysin on sheep erythrocytes, first described by Lovell & Zaki (1966) and subsequently shown by Onon (1979) to be a useful in vitro method of measurement of C. ovis toxin, was employed as described by Onon (1979) as an additional test for the presence of C. ovis toxin in crude filtrates and chromatographic fractions prepared from them.…”

“…The method was as described by Onon (1979), consisting of precipitation by 35-65 0% (NH4)2SO4, dialysis, ion-exchange chromatography on a 9 cm x 2 cm column of Sephadex CM-50 buffered with 0O1 M sodium acetate, pH 6 1. After the unbound material had been eluted, a linear NaCl gradient was applied.…”

“…First, no detailed knowledge of the characteristics of the exotoxin of C. ovis (which it was thought by Petrie & McClean to resemble), became available until the papers of Soucek & Souckova (1971, Carne & Onon (1978) and Onon (1979). These have shown that C. ovis toxin is a basic glycoprotein, MW 14500+1000, which is a phospholipase D; it attacks sphingomyelin to produce ceramide phosphate and choline; when inoculated intradermally or subcutaneously in guinea-pigs, rabbits or sheep it causes marked leakage of plasma from blood and lymph vessels, producing local oedema; it does not produce congestion of the adrenal cortex of the guinea-pig, which is characteristic of diphtheria toxin.…”