Characterization of non-toxic mutant toxins of Vero toxin 1 that were constructed by replacing amino acids in the A subunit.
Three mutant toxins of Vero toxin 1 (VT1) produced by Escherichia coli strains carrying mutant VT1 genes that were constructed by oligonucleotide-directed site-specific mutagenesis were purified to homogeneity. They are E167Q (glutamic acid at position 167 from the N-terminus of the A subunit was replaced by glutamine), R170L (arginine at position 170 from the N-terminus of the A subunit was replaced by leucine) and E167Q-R170L (glutamic acid at position 167 and arginine at position 170 were replaced by glutamine and leucine, respectively). The purified E167Q and E167Q-R170L had markedly decreased activities, such as inhibition of protein synthesis, cytotoxicity to Vero cells and mouse lethality. The decrease in the R170L activities was less than those of the other two mutant VT1s. Neither additive nor synergistic decreases of the activities were observed in the double mutant E167Q-R170L in which two amino acids were replaced. Ouchterlony double gel diffusion showed that antiserum against the purified E167Q-R170L gave a line of identity between mutant VT1s and VT1. Moreover, anti-E167Q-R170L antiserum showed similar activity to that of anti-VT1 antiserum in neutralizing the Vero cell cytotoxicity and mouse lethality of VT1. The data suggest that these mutant VT1s, especially E167Q and E167Q-R170L, may be candidate toxoids that protect against diseases caused by Verocytotoxin-producing E. coli.
Ohmura
M
M
Department of Microbiology, Faculty of Medicine, Kyoto University, Japan.
Yamasaki
S
S
Kurazono
H
H
Kashiwagi
K
K
Igarashi
K
K
Takeda
Y
Y
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity*; D001483:Base Sequence; D002470:Cell Survival; D002853:Chromatography, Liquid; D003603:Cytotoxins / Q000633:toxicity*; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D014176:Protein Biosynthesis; D022622:Shiga Toxin 1; D014121:Toxoids; D014709:Vero Cells
D001427:Bacterial Toxins; D003603:Cytotoxins; D022622:Shiga Toxin 1; D014121:Toxoids