THE ENZIME OROTATE PHOSPHORIBOSYLTRANSFERASE (OPRT) FROM MYCOBACTERIUM TUBERCULOSIS : BIOCHEMICAL CHARACTERIZATION AND GENE KNOCKOUT FOR AN ATTENUATED STRAIN DEVELOPMENT

Ardala Breda (INCTT); Cristopher Zandoná Schneider (INCTT); Luiz Augusto Basso (INCTT); Diógenes Santiago Santos (INCTT)

Tuberculosis (TB) is a chronic infection mainly caused by Mycobacterium tuberculosis (MTB), killing over 2 million people annually. TB resurgence and multi and extensive-drug resistant strains leads to the need of novel TB treatments. The orotate phosphoribosyltransferase (OPRT) catalyses OMP formation, the fifth reaction of the de novo synthesis of pyrimidine nucleotides, an essential pathway for microorganism viability; and is an attractive antitubercular drug target. The aims of this study were: I. Amplification and cloning of the OPRT coding pyrE gene, overexpression and purification of the recombinant enzyme for further biochemical characterization. II. To knockout the pyrE gene, leading to a MTB strain dependent of pyrimidine salvage pathway to suffice its anabolic needs for such nucleotides. The full-length pyrE coding region was amplified from MTB genome and cloned at the expression vector pET-23a+. Resulting plasmid was introduced into the E. coli BL21(DE3) strain and grown in LB medium at 37°C. Best protein expression was obtained after 12h with 1mM IPTG induction. Recombinant OPRT was purified with anion exchange and size exclusion liquid chromatography columns using a FPLC system, with purity higher than 92%. The oligomeric state determination by size exclusion liquid chromatography showed that MTB OPRT is formed by two identical subunits of 21 KDa each. OPRT activity (OMP synthesis) was measured spectrophotometrically for kinetic constants determination, allowing a better understanding of pyrimidine metabolism of mycobacteria. For gene knockout experiments, nucleotide lengths of 1Kb upstream and downstream of pyrE gene were independently amplified from MTB genome and cloned into pCR-Blunt vectors. Both fragments were digested with appropriated enzymes and cloned into a p2NIL vector. Resistance markers were introduced into the p2NIL vector, allowing visual selection of proper knockout constructs plasmids, as well as MTB cells where homology recombination process has occurred. Obtained knockout MTB cells will be grown with uracil supplementation and tested for its infection and immunogenic capabilities. An MTB strain unable to catalyze de novo synthesis of pyrimidines will expected to constitute an attenuated strain, capable of inducing a long-term protective immune response to TB infection.