Construction and analysis of a Vibrio cholerae delta-aminolevulinic acid auxotroph which confers protective immunity in a rabbit model

Unit of Molecular Microbiology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
Infection and Immunity (Impact Factor: 3.73). 07/1992; 60(6):2188-93.
Source: PubMed


Vibrio cholerae CVD101 is a very effective live vaccine. Although this strain does not produce active cholera toxin because of a mutation in the gene for the cholera toxin A subunit, it still shows residual pathogenicity. To attenuate CVD101 further, we set out to isolate derivatives of CVD101 which were limited in their ability to proliferate in vivo. Two delta-aminolevulinic acid auxotrophs of CVD101, designated V286 and V287, were isolated by transposon mutagenesis and penicillin enrichment. Southern blotting revealed that the mutants differed with respect to the location of the transposon insertion. Under aerobic conditions, in the absence of delta-aminolevulinic acid, both mutants showed diminished growth compared with CVD101. The growth of V286 was most severely affected. Microaerophilic growth of both mutants was less affected. Competition experiments with a rabbit model showed that strain V286 was found in numbers 10(3)- to 10(4)-fold lower than its parental strain. This observation indicates that strain V286 is impaired in its ability to colonize the rabbit intestine. It also supports an important role for aerobic growth in the colonization of the intestine by V. cholerae. Vaccination of rabbits with a single dose of strain V286 resulted in full protection against challenge with a virulent strain. Strain V286 was not shed from rabbits in a cultivatable form. Our results suggest that delta-aminolevulinic acid auxotrophy can attenuate V. cholerae by limiting its ability to colonize without affecting its capacity to induce protective immunity. Furthermore, this type of mutation may prevent the spread of V. cholerae vaccine strains in the environment.


Available from: Elisabeth Margaretha Bik
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    • "It has ability to deliver covalently attached antigens to the mucosal cells via binding to GM1 ganglioside receptor on the surface of epithelial cells [19, 20]. CTB was used as an adjuvant to increase the immune response level in many different vaccines specially vaccines against toxigenic Vibrio cholerae strains [21-23]. Many efforts have been performed to produce CTB in E. coli [24], Lactobacillus and Bacillus brevis [25, 26] or even Vibrio cholerae strains lacking the CTA gene [7]. "
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    ABSTRACT: Background: Cholera toxin B subunit (CTB) has been extensively considered as an immunogenic and adjuvant protein, but its yield of expression is not satisfactory in many studies. The aim of this study was to compare the expression of native and mutant recombinant CTB (rCTB) in pQE vector. Methods: ctxB fragment from Vibrio cholerae O1 ATCC14035 containing the substitution of mutant ctxB for amino acid S128T was amplified by PCR and cloned in pGETM-T easy vector. It was then transformed to E. coli Top 10F' and cultured on LB agar plate containing ampicillin. Sequence analysis confirmed the mature ctxB gene sequence and the mutant one in both constructs which were further subcloned to pQE-30 vector. Both constructs were subsequently transformed to E. coli M15 (pREP4) for expression of mature and mutant rCTB. Results: SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 hours after induction and Western-blot analysis confirmed the presence of rCTB in blotting membranes. The expression of mutant rCTB was much higher than mature rCTB, which may be the result of serine-to-threonine substitution at position 128 of mature rCTB amino acid sequence created by PCR mutagenesis. The mutant rCTB retained pentameric stability and its ability to bind to anti- cholera toxin IgG antibodies. Conclusion: Point mutation in ctxB sequence resulted in over-expression of rCTB, probably due to the increase of solubility of produced rCTB. Consequently, this expression system can be used to produce rCTB in high yield.
    Iranian biomedical journal 07/2014; 18(3):130-5. DOI:10.6091/ibj.1165.2014
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    • "Root-colonizing ability is not only important in biocontrol , but can also be predicted to be essential in other applications of microbial inoculants of seeds, such as for biofertilization, phytostimulation and phytoremediation, in which the effectiveness of the inoculant is likely to be dependent on the establishment of a minimum population size. It is also worthy of mention that many colonization traits important for biocontrol of plant diseases also play a role in colonization of animal tissues by bacteria (Drake & Montie, 1988; Weiser et al., 1990; Rijpkema et al., 1992; Chiang & Mekalanos, 1998; Lugtenberg & Dekkers, 1999). Phenazine derivatives not only kill fungi but the phenazine derivative pyocyanin, produced by certain Pseudomonas aeruginosa strains, is also involved in killing of animal cells and tissues (Mahajan et al., 1999). "
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    New Phytologist 02/2003; 157(3):503 - 523. DOI:10.1046/j.1469-8137.2003.00686.x · 7.67 Impact Factor
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    • "The three colonization traits shown here to be important for biocontrol of the plant disease tomato root and foot rot also play a role in the colonization of animal tissues by bacteria (Chiang and Mekalanos 1998; Drake and Montie 1988; Lugtenberg and Dekkers 1999; Rijpkema et al. 1992; Weiser et al. 1990). Phenazine derivatives do not only kill fungi, but the phenazine-derivative pyocyanine, produced by P. aeruginosa, is also involved in killing animal cells and tissues (Mahajan et al. 1999). "
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