Resumo

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, infects one-third of the world population. The World Health Organization estimates that 8.7 million new TB cases occurred in 2011, resulting in 1.4 million deaths worldwide. There is a critical need for the development of new drugs to control TB worldwide. Enzymes involved in purine and pyrimidine biosynthesis have important roles in cellular metabolism, as they provide nucleotides that are essential components of a number of biomolecules. Guanosine 5’-monophosphate (GMP) synthase (EC 6.3.5.2), encoded by guaA gene (Rv3396c), is a key enzyme in both purine de novo synthesis and salvage pathways of guanine nucleotides that catalyzes the conversion of xanthosine 5’-monophosphate (XMP) into GMP. The goals of this work are to knockout guaA gene from M. tuberculosis and to validate GMP synthase as a molecular target for drug candidates to treat human TB. Genomic region including guaA gene (1.6 kb) plus about 500 bp flanking each 5’ and 3’ regions was obtained by PCR from M. tuberculosis H37Rv genomic DNA and cloned into pUC19 plasmid. Target gene was disrupted by the insertion of a kanamycin cassette and subcloned into pPR27xylE vector. M. tuberculosis H37Rv strain was transformed with pPR27xylE::guaA 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 with or without 20 µg/mL guanine, and cultivated at 39°C, because of counter selective properties of sacB gene and thermosensitive origin of replication from pPR27xylE plasmid. Colonies were almost all yellow when tested with catechol either on plates containing guanine or not. The few white colonies obtained were checked by PCR and none were mutants for guaA gene, which is an indicative of the essentiality of this gene under the employed experimental conditions. Complementation experiment with guaA gene from M. smegmatis and synthesis of GMP synthase inhibitors are under way. These experiments will confirm guaA essentiality and validate this gene as a molecular target for drug design. GMP synthase inhibitors might prove to be useful for future development of a new drug to treat human TB.