Role of alpha-toxin in Clostridium perfringens infection determined by using recombinants of C. perfringens and Bacillus subtilis

Abstract

Clostridium perfringens type A strains which differed in alpha-toxin (phospholipase C [PLC]) productivity were inoculated intraperitoneally or intravenously into mice, and then their 50%o mouse lethal doses (LD50) were determined. Strain NCTC 8237 produced ninefold higher PLC activity than strain 13. The mean LD50 for the former was 1 log unit lower than that for the latter. Two isogenic strains were constructed from strain 13: strain 13(pJIR418a) (pJIR418a contains the plc gene), which produced ninefold higher PLC activity than strain 13; and strain 13 PLC-, which showed no PLC productivity at all because of transformation-mediated gene disruption. The mean LD50 for strain 13(pJIR418a) was 1 log unit lower than those for strain 13 PLCand strain 13. These results indicate that PLC functions as a virulence-determining factor when it is produced in a sufficient amount. Such a difference in LDso was also observed between Bacillus subtilis with and without the cloned plc gene. Inoculation of B. subtilis PLC' intravenously into mice caused marked thrombocytopenia and leukocytosis. Mice inoculated with B. subtilis at 2 LD50 died because of circulatory collapse. Histological examination revealed that intravascular coagulation and vascular congestion occurred most prominently in the lungs. These results suggest that PLC plays a key role in the systemic intoxication of clostridial myonecrosis, probably by affecting the functions of platelets and phagocytes.

The hydA gene encoding the H2-evolving hydrogenase of Clostridium perfringens: molecular characterization and expression of the gene

“…C. perfringens strains used in this study were type A strains: NCTC8237, 13, a transformation-competent strain [10], and its derivative, 13Plc 3 [11]. They were grown at 37³C in TYG medium [12], consisting of 3 g tryptone, 2 g yeast extract, 0.1 g sodium thioglycolate and 5 g l 31 glucose.…”

“…AB016820). Disruption of the hydA gene in the C. perfringens strain 13 by a Campbell-like insertion event was performed in the same manner as described previously [11], except for a vector, a derivative of pJIR418D [11], which contained a 0.89-kb EcoRI-BamHI fragment within the hydA gene of strain 13 at the multiple cloning sites ( Fig. 1b).…”

“…To inhibit a possible revertant strain, strain 13HydA 3 was grown in TYG medium containing chloramphenicol (10 Wg ml 31 ). Strain 13Plc 3 [11], which was the same as strain 13HydA 3 except for a disrupted gene, was used as a control. Their growth rates and ¢nal cell densities were similar to the values described above, although slightly lowered by the antibiotic (data not shown).…”

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

“…Although long suspected, it is now clear that the ␣-toxin, the principal virulence factor produced by type A strains of C. perfringens, plays the major role in the disease (McDonel, 1986). Recent virulence studies, in which mice were infected with C. perfringens mutants unable to produce various toxins, demonstrated unambiguously the devastating effect of ␣-toxin in gross inflammation, haematuria, tissue invasion, tissue damage and necrosis (Awad et al, 1995;Ninomiya et al, 1994). This broad spectrum of pathogenic activities reflects the multifunctional nature of the ␣-toxin, encoded by the plc gene, for it is endowed with both phospholipase C (lecithinase) and sphingomyelinase activities and functions as a powerful cytolysin hydrolysing phospholipids in the cell membranes of erythrocytes, phagocytes and fibroblasts (Titball, 1993).…”

A Clostridium perfringens hem Gene Cluster Contains a cysGB Homologue That Is Involved in Cobalamin Biosynthesis

“…examination of nucleotide sequence deviation: three type-A strains, PB6K (40), KZ211 (16) and 8-6 (32); a type-B strain, NCIB10691; a type-C strain, NCIB 10662; two type-D strains, L9 (47) and NCIB10663; and a type-E strain, NCIB 10748. An E. coli-C. perfringens shuttle vector, pJIR418 (39), and its derivative, pJIR418D (26), were used for the construction of a cysGB mutant strain by homologous recombination. C. perfringens strain 13 Plc , of which the phospholipase C gene was disrupted by homologous recombination (26), was used as a control for strain 13 CysGB .…”