• Mycobacterium tuberculosis
• oxidative stress
• TbD1
• virulence

Mycobacterium tuberculosis and Mycobacterium africanum strains, the etiologic agents of human tuberculosis, have been classified in different phylogenetic lineages on the basis of their spoligotype (lineages 1-4 and 7 for M. tuberculosis, and lineages 5-6 for M. africanum). M. tuberculosis strains belonging to lineages 1 and 7, and M. africanum lineages 5 and 6 share a conserved 2153-bp genomic region called TbD1 (M. tuberculosis specific deletion 1). This region is also conserved in other mycobacterial species phylogenetically more closely related to the hypothetical ancestral progenitor, such as Mycobacterium canettii, Mycobacterium bovis or other animal-adapted mycobacterial species. In contrast, the TbD1 region is deleted in the so-called M. tuberculosis “modern” strains, belonging to lineages 2, 3 and 4. Interestingly, while M. tuberculosis strains harbouring an intact TbD1 region (“ancestral” strains) are restricted to specific geographic areas, such as countries bordering the Indian Ocean (lineage 1) or the Horn of Africa (lineage 7), “modern” strains are spread worldwide and account for the most cases of human tuberculosis. In this study, a panel of M. tuberculosis strains belonging to different lineages were tested by PCR for the presence of an intact TbD1 locus and for their resistance/survival after in vitro exposure to stresses mimicking the intracellular environment during active phases of infection. Stress conditions included i) the exposure to reactive oxygen intermediates (ROI), such as hydrogen peroxyde, cumene hydroperoxyde and plumbagine; ii) exposure to reactive nitrogen intermediates (RNI), including sodium nitroprusside and NaNO2; iii) acidic pH. Ancestral M. tuberculosis strains resulted more sensitive than the modern strain M. tuberculosis H37Rv to ROI, while no differences were observed among the strains in response to low pH and RNI. To assess the role of the TbD1 genomic region in response to oxidative stress, a mutant M. tuberculosis ancestral strain (M. tuberculosis 79112) deleted for the TbD1 region was constructed (M. tuberculosis 79112 ∆TbD1). This strain was obtained using the “recombineering” strategy, an effective approach in which phage-encoded recombination protein (Che9c), expressed under control of an inducible promoter, allows recombination to occur at high frequency, thus facilitating allelic exchange using linear DNA substrates. The correct replacement of the TbD1 region with a kanamycin-resistance cassette in the M. tuberculosis 79112 ∆TbD1 strain was confirmed by PCR analysis. Moreover, a complemented strain (M. tuberculosis 79112 ∆TbD1::2G12), in which an intact TbD1 locus was integrated into the genome of the mutant strain, was successfully constructed. The analysis of survival of mutant and wild-type ancestral strains after exposure to ROI in vitro will contribute to clarify the impact of TbD1 in the resistance of M. tuberculosis to oxidative stress.