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Research Article

Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.

C C Häse, L S Thai, M Boesman-Finkelstein, V L Mar, W N Burnette, H R Kaslow, L A Stevens, J Moss, R A Finkelstein
C C Häse
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L S Thai
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M Boesman-Finkelstein
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V L Mar
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W N Burnette
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H R Kaslow
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L A Stevens
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J Moss
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R A Finkelstein
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ABSTRACT

The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins. V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination. The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)

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Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.
C C Häse, L S Thai, M Boesman-Finkelstein, V L Mar, W N Burnette, H R Kaslow, L A Stevens, J Moss, R A Finkelstein
Infection and Immunity Aug 1994, 62 (8) 3051-3057; DOI:

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Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs.
C C Häse, L S Thai, M Boesman-Finkelstein, V L Mar, W N Burnette, H R Kaslow, L A Stevens, J Moss, R A Finkelstein
Infection and Immunity Aug 1994, 62 (8) 3051-3057; DOI:
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