Purification and characterization of novel toxin produced by Vibrio cholerae O1.
Vibrio cholerae WO7 (serogroup O1) isolated from patients with diarrhea produces an extracellular toxin despite the absence of ctx, zot, and ace genes from its genome. The toxin elongates Chinese hamster ovary cells, produces fluid accumulation in ligated rabbit ileal loops, and agglutinates freshly isolated rabbit erythrocytes. Maximal production of this toxin (WO7 toxin) was seen in AKI medium with the pH adjusted to 8.5 at 37 degrees C under shaking conditions. We purified this toxin to homogeneity by sequential ammonium sulfate precipitation, affinity chromatography using a fetuin-Sepharose CL-4B column, and gel filtration chromatography, which increased the specific activity of the toxin by 1.6 x 10(6)-fold. The toxin is heat labile and sensitive to proteases and has a subunit structure consisting of two subunits with molecular masses of about 58 and 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Agglutination of GM1-coated sheep erythrocytes by toxin suggests that GM1 might be the physiologic receptor for WO7 toxin on the enterocytes. An immunodiffusion test between the antiserum raised against the purified WO7 toxin and the purified toxin gave a well-defined precipitation band. In the immunoblot assay, two bands were observed in the 58- and 40-kDa region. At the same time, antiserum against WO7 toxin failed to show any cross-reactivity with cholera toxin or Escherichia coli heat-labile toxin (LT1) in an immunodiffusion test or immunoblot assay. The enterotoxic activity of WO7 toxin could be inhibited by antiserum against purified WO7 toxin. Our results indicate that WO7 toxin is structurally and functionally distinct from other cholera toxins and that the enterotoxic activities expressed by WO7 toxin appear to contribute to the pathogenesis of disease associated with V. cholerae O1 strains.
Walia
K
K
Department of Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Ghosh
S
S
Singh
H
H
Nair
G B
GB
Ghosh
A
A
Sahni
G
G
Vohra
H
H
Ganguly
N K
NK
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002522:Chlorocebus aethiops; D002772:Cholera Toxin / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006224:Cricetinae; D008969:Molecular Sequence Data; D011817:Rabbits; D014709:Vero Cells; D014734:Vibrio cholerae / Q000472:pathogenicity*
D002772:Cholera Toxin