title: Identification and characterization of Clostridium perfringens beta toxin variants with differing trypsin sensitivity and in vitro cytotoxicity activity.
abstract: By producing toxins, Clostridium perfringens causes devastating diseases of both humans and animals. C. perfringens beta toxin (CPB) is the major virulence determinant for type C infections and is also implicated in type B infections, but little is known about the CPB structure-function relationship. Amino acid sequence comparisons of the CPBs made by 8 randomly selected isolates identified two natural variant toxins with four conserved amino acid changes, including a switch of E to K at position 168 (E168K) that introduces a potential trypsin cleavage site into the CPB protein of strain JGS1076. To investigate whether this potential trypsin cleavage site affects sensitivity to trypsin, a primary host defense against this toxin, the two CPB variants were assayed for their trypsin sensitivity. The results demonstrated a significant difference in trypsin sensitivity, which was linked to the E168K switch by using site-directed recombinant CPB (rCPB) mutants. The natural CPB variants also displayed significant differences in their cytotoxicity to human endothelial cells. This cytotoxicity difference was mainly attributable to increased host cell binding rather than the ability to oligomerize or form functional pores. Using rCPB site-directed mutants, differences in cytotoxicity and host cell binding were linked to an A300V amino acid substitution in the strain JGS1076 CPB variant that possessed more cytotoxic activity. Mapping of sequence variations on a CPB structure modeled using related toxins suggests that the E168K substitution is surface localized and so can interact with trypsin and that the A300V substitution is located in a putative binding domain of the CPB toxin.
authors: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors: James R
authors: JR
authors: Theoret
authors: California Animal Health and Food Safety Laboratory, San Bernardino Branch, School of Veterinary Medicine, University of California-Davis, San Bernardino, California, USA.
authors: Francisco A
authors: FA
authors: Uzal
authors: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA bamcc@pitt.edu.
authors: Bruce A
authors: BA
authors: McClane
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D003016:Clostridium perfringens / Q000472:pathogenicity*; D014644:Genetic Variation; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D052899:Pore Forming Cytotoxic Proteins / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D014357:Trypsin / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
chemical_list: D001427:Bacterial Toxins; C031884:CPB protein, Clostridium perfringens; D052899:Pore Forming Cytotoxic Proteins; D037521:Virulence Factors; D014357:Trypsin
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