Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.
Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.
Alape-Girón
A
A
Microbiology and Tumorbiology Center, Karolinska Institutet, Stockholm, Sweden.
Flores-Díaz
M
M
Guillouard
I
I
Naylor
C E
CE
Titball
R W
RW
Rucavado
A
A
Lomonte
B
B
Basak
A K
AK
Gutiérrez
J M
JM
Cole
S T
ST
Thelestam
M
M
D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology / Q000472:pathogenicity*; D006224:Cricetinae; D003412:Cricetulus; D004912:Erythrocytes / Q000187:drug effects; D005738:Gas Gangrene / Q000382:microbiology*; D006461:Hemolysis; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000633:toxicity; D012756:Sheep; D010738:Type C Phospholipases / Q000737:chemistry* / Q000633:toxicity*; D014774:Virulence
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D010738:Type C Phospholipases