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Journal of Bacteriology, July 2001, p. 4024-4032, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.4024-4032.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis

Michael G. Jobling and Randall K. Holmes*

Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80220

Received 11 December 2000/Accepted 27 March 2001

Cholera toxin (CT) is the prototype for the Vibrio cholerae-Escherichia coli family of heat-labile enterotoxins having an AB5 structure. By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties. Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin. Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68+V72). In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity. For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10- and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12- to 50-fold more than the reduction in toxicity. These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use.

* Corresponding author. Mailing address: Department of Microbiology, B175, University of Colorado Health Sciences Center, 4200 East 9th Ave., Denver, CO 80220. Phone: (303) 315-7903. Fax: (303) 315-6785. E-mail: Randall.Holmes{at}UCHSC.edu.

Journal of Bacteriology, July 2001, p. 4024-4032, Vol. 183, No. 13
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.13.4024-4032.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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