Immunization with chemically detoxified pertussis toxin can prevent severe whooping cough with an efficacy similar to that of the cellular pertussis vaccine, which normally gives unwanted side effects. To avoid the reversion to toxicity and the loss of immunogenicity that may follow chemical treatment of pertussis toxin, inactive toxins were constructed by genetic manipulation. A number of genetically engineered alleles of the pertussis toxin genes, constructed by replacing either one or two key amino acids within the enzymatically active S1 subunit, were introduced into the chromosome of strains of Bordetella pertussis, B. parapertussis, and B. bronchiseptica. These strains produce mutant pertussis toxin molecules that are nontoxic and immunogenic and that protect mice from the intracerebral challenge with virulent Bordetella pertussis. Such molecules are ideal for the development of new and safer vaccines against whooping cough.
SummaryIt has been shown that resting cells of Agrobacteriurn radiobacter catalyze a sequence of two stereospecific hydrolytic reactions leading to the complete transformation of racemic hydantoins to D-amino acids. This report describes some properties of this new biocatalyst and its potential application for the production of some D-amino acids, which are used as intermediates in the preparation of semisynthetic penicillins and cephalosporins.
We have attempted to express the Helicobacter pylori vacuolating cytotoxin in Escherichia coli. Although the 95-kDa VacA polypeptide was expressed abundantly, it completely lacked any biological activity. In addition, this material failed to induce neutralizing antibodies after immunization of rabbits. In contrast, highly purified high-molecular-mass cytotoxin from the supernatant of H. pylori cultures was active in a HeLa cell assay and effectively induced a neutralizing response in rabbits. Neutralizing sera were shown to contain a high proportion of antibodies which recognized conformational epitopes found only on the native toxin. The data indicate that toxin-neutralizing epitopes are conformational and that potential vaccines based on the cytotoxin may benefit from the use of the intact molecule.
We show that solid and liquid media, supplemented only with cyclodextrins and free of blood and its derivatives, support the growth of Helicobacter pyloyi. These media can be used for primary isolation of the bacteria from biopsy samples, routine laboratory growth, and large-scale industrial fermentation.
Using computer modelling, we have identified some of the residues of the A subunit of cholera toxin (CT) and heat-labile toxin that are involved in NAD binding, catalysis, and toxicity. Here we describe the site-directed mutagenesis of the CT gene and the construction of CT mutants. Nine mutations of the A subunit gene were generated. Six of them encoded proteins that were fully assembled in the AB 5 structure and were nontoxic; these proteins were CT-D53 (Val-533Asp), CT-K63 (Ser-633Lys), CT-K97 (Val-973Lys), CT-K104 (Tyr-1043Lys), CT-S106 (Pro-1063Ser), and the double mutant CT-D53/K63 (Val-533Asp, Ser-633Lys). Two of the mutations encoded proteins that were assembled into the AB 5 structure but were still toxic; these proteins were CT-H54 (Arg-543His) and CT-N107 (His-1073Asn). Finally, one of the mutant proteins, CT-E114 (Ser-1143Glu), was unable to assemble the A and the B subunits and produced only the B oligomer. The six nontoxic mutants were purified from the culture supernatants of recombinant Vibrio cholerae strains and further characterized. The CT-K63 mutant, which was the most efficient in assembly of the AB 5 structure, was used to immunize rabbits and was shown to be able to induce neutralizing antibodies against both the A and B subunits. This molecule may be useful for the construction of improved vaccines against cholera.
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