Resumo

Human tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, remains a leading cause of mortality worldwide. Thus, there is a continuous need to find promising molecular targets for the development of anti-TB agents and to identify virulence determinants associated with M. tuberculosis pathogeneses aiming the obtainment of attenuated mutant strains as new vaccine candidates against human TB. Cytidine deaminase, encoded by cdd gene (Rv3315c), is a pyrimidine salvage pathway enzyme that recycles cytidine and 2’-deoxycytidine to uridine and 2’-deoxyuridine synthesis, respectively. This work aims to construct and characterize a M. tuberculosis mutant strain for cdd gene to validate its biological importance in mycobacteria metabolism, persistence, and virulence. Genomic region including cdd gene (402 bp) plus about 800 bp upstream and 600 bp downstream regions was obtained by PCR from M. tuberculosis H37Rv DNA genomic and cloned into pUC19 plasmid. Target gene was disrupted by the insertion of a kanamycin cassette and then subcloned into pPR27xylE vector. The ortholog cdd gene from Mycobacterium smegmatis was cloned into pNIP40 vector, which is being used to obtain the complemented strain. M. tuberculosis H37Rv strain was transformed with pPR27xylE::cdd kan construction and kanamycin was used to select transformants on plates. Catechol was dropped on colonies to select the ones that contain the plasmid which turned yellow since pPR27xylE vector contains counter selective properties of the xylE gene. Yellow colonies were cultivated in liquid medium with antibiotic at 32°C. Individual cultures were plated on solid medium containing kanamycin, 2% sucrose, and cultivated at 39°C, because of counter selective properties of sacB gene and thermosensitive origin of replication from pPR27xylE plasmid. Colonies were observed after 4 weeks and all big colonies were white when tested with catechol. Nine white colonies were checked by PCR and four clones had the kanamycin cassette inserted, which is an indicative that cdd is not an essential gene under the employed experimental conditions. Experiments for the construction of complemented strain, in vitro growth studies and mice infection are under way to analyze the biological importance of cdd gene in M. tuberculosis metabolism and the mutant strain attenuation. M. tuberculosis mutant for cdd gene might be an attenuated strain useful for future development of a new vaccine candidate against TB.