Speculation and C₂ Toxin Production by Clostridium botulinum Type C Strains Producing No C₁ Toxin

Abstract

All of the 8 strains that were previously assumed to be nontoxigenicClostridium botulinum type C were re-examined for their toxigenicity and were demonstrated by trypsinization of the culture filtrates to produce C2 toxin under improved cultural conditions.One per cent glucose added to trypticase peptone medium enhanced C2 toxin production.The larger the spore population, the higher the C2 toxicity and when spore population was smaller than a level of 104/ml, no C2 toxicity was demonstrated.The C2 toxin was produced only during sporulation and not during vegetative growth.Serikawa et al (5) reported nontoxigenic Clostridium botulinum type C strains identified by agglutination in addition to the routine tests for cultural and biochemical properties. Eklund and Poysky (1) reported that some strains of C. botulinum type C producing no dominant C1 toxin produced C2 toxin, which often required treatment with trypsin before its toxicity could be demonstrated. On the basis of their findings, we tested again the nontoxigenic strains we isolated , and found the production of C2 toxin, which was neutralized by antiserum homologous to anti-C2 toxin prepared by Eklund and Poysky (1). It was also found that C2 toxin production was closely related to sporulation. This report describes the production of C2 toxin and sporulation by these strains. MATERIALS AND METHODSStrains. Eight isolates of C. botulinum type C, producing no Ci component of type C toxin (5), 85-14, 85-18, 86-16, 86-18, 86-19, 92-13, 98-1, and 98-2, from soil samples were used.Toxin production. Pre-reduced TYG medium consisted of 3% trypticase (BBL), 2% yeast extract (Difco), 1% glucose, 0.4% Na2CO3, 0.045% K2HPO4, 0.045% KH2PO4, 0.09% (NH4)2SO4, 0.09% NaCl, 0.009% MgSO4-7H2O, 0.009% CaCl2. 2H2O, 0.05% L-cysteine-HCl, and 0.0001% resazulin. Percentage is expressed as weight per volume unless otherwise is stated. A 0.2-ml volume of a bacterial culture grown overnight in liver broth was transferred into an 8-ml volume of pre-591

Binary Bacterial Toxins: Biochemistry, Biology, and Applications of CommonClostridiumandBacillusProteins

“…Each "A" and "B" component of the Clostridium and Bacillus binary toxins, but not those from C2, is respectively synthesized with a signal peptide consisting of 29 to 49 and 39 to 47 residues (331). These findings are consistent with proteins secreted during logarithmic growth; however, the C2 toxin is evident from sporulating C. botulinum only after sporangium lysis (289,331). It was recently shown that an extracellular chaperone protein, PrsA, is important for efficient folding and secretion of PA83 from Bacillus species via a Sec-dependent route (477), but it is unknown if similar chaperones play any role with binary toxin components produced by the other bacilli.…”

“…However, the binary C2 enterotoxin produced by toxin types C and D is devoid of neurotoxicity but implicated in a fatal enteric disease of waterfowl, is cytopathic for many different cell types, and induces vascular permeability, necrotic-hemorrhagic lesions, and a lethal fluid accumulation in the lungs and intestinal tracts of various animals (113,189,191,221,276,297,298,300,302,303,(305)(306)(307)(308)390). C2 toxin is synthesized by C. botulinum during sporulation (289) and incorporated into the spore coat (481), which is akin to the single-chain enterotoxin of C. perfringens or protective antigen from B. anthracis edema and lethal toxins (93,130,364,371,465,466). Much of the pioneering work on characterizing the cell binding and translocation component (C2II), as well as the enzyme component (C2I), of C2 toxin was initiated by Ohishi and coworkers throughout the 1980s.…”

Isolation of Clostridium difficile from the Feces and the Antibody in Sera of Young and Elderly Adults

“…Serial 10-fold dilutions were prepared in the dilution solution previously described (16). Samples of 0.1 ml of each serial dilution (10-1 to were plated onto CCFA (5) modified by the addition of 300 I'g of cycloserine (Sigma Chemical Co., St. Louis, Mo., U.S.A.) per ml, 10 I'g of cefoxitin (Merck Sharp & Dohme Research Laboratory, West Point, Pa., U.S.A.) per ml, and l0l'g of lysozyme (Seikagaku Kogyo, Tokyo) per ml, which had been stored in an anaerobic jar under hydrogen gas for 18 to 24 hr before use.…”

General Biology and Physiology

“…Toxin expression appears to be maximal in stationary phase, and the highest levels of toxin production are generally found in cell populations which undergo rapid autolysis and do not sporulate [428,429]. Only in the case of type C 2 toxin has an association between toxin production and sporulation been reported [430].…”