RESUMO
Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP), encoded by deoD gene (Rv3307), is an enzyme from the purine salvage pathway, which has been widely studied as a molecular target for the development of inhibitors with potential antimycobacterial activity. However, the role of MtPNP in tuberculosis pathogenesis and dormancy is still unknown. The present work aims to construct a deoD knockout strain from M. tuberculosis, to evaluate the role of MtPNP in the growth of M. tuberculosis under oxygenated condition and in a dormancy model, and to assess whether deoD gene is important for M. tuberculosis invasion and growth in macrophages. The construction of a knockout strain for deoD gene was confirmed at DNA level by PCR and protein level by Western blot and LC-MS/MS. The deoD gene is not required for M. tuberculosis growth and survival under oxygenated and hypoxic conditions. The disruption of deoD gene did not affect mycobacterial ability to invade and grow in RAW 264.7â¯cells under the experimental conditions employed here.
Assuntos
Macrófagos/microbiologia , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/fisiologia , Animais , Sequência de Bases , Cromatografia Líquida , DNA Bacteriano/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Bacterianos/genética , Camundongos , Mycobacterium tuberculosis/patogenicidade , Oxigênio/metabolismo , Células RAW 264.7 , Espectrometria de Massas em Tandem , Tuberculose/microbiologiaRESUMO
Para-aminosalicylic acid (PAS) is an antibiotic that was largely used for the multi-therapy of tuberculosis in the twentieth century. To try to overcome the inconvenience of its low efficacy and poor tolerance, we searched for novel chemical entities able to synergize with PAS using a combination screening against growing axenic Mycobacterium tuberculosis. The screening was performed at a sub-inhibitory concentration of PAS on a library of about 100,000 small molecules. Selected hit compounds were analyzed by dose-response and further probed with an intracellular macrophage assay. Scaffolds with potential additive effect with PAS are reported, opening interesting prospects for mechanism of action studies. We also report here evidence of a yet unknown bio-activation mechanism, involving activation of pyrido[1,2-a]pyrimidin-4-one (PP) derivatives through the Rv3087 protein.