CONSTRUCTION AND CHARACTERIZATION OF RECOMBINANT VIBRIO-CHOLERAE STRAINS PRODUCING INACTIVE CHOLERA-TOXIN ANALOGS

The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribo sylating 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 previousl y been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both A rg-7 and Glu-112, each of which has been shown to produce subunits lac king ADP-ribosyltransferase activity. The mutated gene fragment was ex changed for the wild-type copy in the previously cloned ctxAB operon f rom Fl Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, tot, was inac tivated by an insertional mutation to create the new plasmid construct pCT2. Additionally, a DNA fragment encoding the B subunit of CT-1 (C T produced by classical biotype, Inaba serotype V. cholerae strain 569 B) was exchanged for the homologous part in pCT-2, resulting in the c reation of pCT-1. These plasmid constructs were introduced into the C T-negative V. cholerae mutant strain JBK70 (El Tor biotype, Inaba sero type); CT-A(-)B(+) derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; Fl Tor bi otype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 fro m the current epidemic in India. Recombinant toxins (CT-1 and CT-2*), partially purified from culture supernatants of transformed JBK70, we re shown to be inactive on mouse Y1 adrenal tumor cells and in an in v itro ADP-ribosyltransferase assay. CT-1 and CT-2* reacted with polycl onal and monoclonal antibodies against both A and B subunits of CT. Th e toxin analogs reacted with antibodies against CT-A and CT-B on cellu lose acetate strips and in a G(M1) enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin ban ds with G(M1)-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies we re weaker than the reactions with wild-type holotoxins, V. cholerae st rains carrying ctxA, with either ctxB-1 or ctxB-2, and inactivated to t genes were created by homologous recombination, The recombinant stra ins and the purified toxin analogs were inactive in the infant rabbit animal model. These strains may have use as attenuated live vaccines; the analog toxins themselves might have important applications in conj ugate vaccines as well as in structure-function studies.