Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A

• Published in: Vaccine Vol. 14; no. 16; pp. 1517 - 1522
• Main Authors: Robert, Alma, Silva, Anisia, Benitez, Jorge A., Rodriguez, Boris L., Fando, Rafael, Campos, Javier, Sengupta, Dilip K., Boesman-Finkelstein, Mary, Finkelstein, Richard A.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.11.1996; Elsevier
• Subjects: Animals; Bacterial Vaccines - genetics; Bacterial Vaccines - immunology; Biological and medical sciences; Biotechnology; Cellulase - biosynthesis; Cellulase - genetics; Clostridium - enzymology; Clostridium - genetics; Clostridium thermocellum; DNA Damage; endoglucanase A; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Genetic Vectors - immunology; Genetic Vectors - metabolism; hemagglutinin/protease; Hemagglutinins - genetics; in vivo marker exchange; Metalloendopeptidases - genetics; Methods. Procedures. Technologies; Mice; Microbiology; reporter gene; vaccine; Vaccines, antisera, therapeutical immunoglobulins and monoclonal antibodies; Vaccines, Synthetic - immunology; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Vibrio cholerae; Vibrio cholerae - immunology; Virology; vaccine; hemagglutinin/protease; endoglucanase A; reporter gene; in vivo marker exchange; Vibrio cholerae; Enzyme; Hemagglutinin; Rodentia; Vaccine; Peptidases; Vertebrata; Mammalia; Reporter gene; Immunogenicity; Mouse; Glycosidases; Insertion mutation; Tagging; Bacteria; Hydrolases; Vibrionaceae; Vibrio cholerae elTor
• ISSN: 0264-410X; 1873-2518
• DOI: 10.1016/S0264-410X(96)00105-3

The celA gene encoding Clostridium thermocellum endoglucanase A was expressed in Vibrio cholerae on its own promoter and used to tag a candidate El Tor biotype cholera vaccine strain. Colonies of the tagged strain could be unequivocally distinguished by overlaying them with CM-cellulose indicator agar and Congo Red staining. Expression of celA did not affect growth of V. cholerae in vitro and in vivo. The celA gene was inserted in the chromosomal hap locus encoding V. cholerae hemagglutinin/protease, a putative “detachase”, to create a hap − mutant that could be identified and scored by its halo of cellulolytic activity. The inactivation of hap had a positive effect on colonization in the infant mice model. The above results indicate that celA is a suitable marker gene for V. cholerae and hap is an appropriate locus for insertion of foreign DNA in vaccine development. Inactivation of hap, by increasing the duration of adherence, might decrease excretion of the resulting vaccine vector strain and thus increase its immunogenicity.