Open AccessImproved cytotoxic effects of S almonella ‐producing cytosine deaminase in tumour cells
Author(s) -
MesaPereira Beatriz,
Medina Carlos,
Camacho Eva María,
Flores Amando,
Santero Eduardo
Publication year - 2015
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.12153
Subject(s) - cytosine deaminase , cytotoxic t cell , cytosine , chemistry , biology , microbiology and biotechnology , biochemistry , gene , in vitro , genetic enhancement
Summary In order to increase the cytotoxic activity of a S almonella strain carrying a salicylate‐inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the E scherichia coli codA gene cloned under the control of the P m promoter have been improved by using the T 7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG . Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5‐fluorocytosine, a 5‐fluorouracyl resistant S almonella strain has been constructed by deleting its upp gene sequence. This new S almonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic S almonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different S almonella strains in tumour cell cultures.