Expression of Resuscitation-Promoting Factor C Stimulates the Growth of Mycobacterium bovis BCG and Delays DevR Regulon Activation in Hypoxia.

Abstract

Latent tuberculosis is characterized by the presence of dormant, nonreplicating (DNR) bacilli for years without causing clinical signs and symptoms, remaining as a major reservoir for active tuberculosis. The mechanism through which M. tuberculosis transits from DNR to active bacilli remains unclear. However, resuscitation-promoting factors (Rpfs) could participate in the reactivation. Using recombinant M. bovis BCG that expresses rpfC (M. bovis BCG-pMV261::rpfC), we evaluated the role of RpfC in the growth of bacilli and the expression of 11 hypoxia-regulated genes in comparison with M. bovis BCG-pMV261. The strains were grown in normoxic (21% O₂), hypoxic (5% O₂), and anoxic (< 0.1% O₂) conditions. In normoxic culture, M. bovis BCG-pMV261::rpfC displays a lower expression of sigB and fdxA. In anoxic culture, we did not observe drastic changes in the gene expression, except for those involved in electron transport during anaerobic respiration (pdxA, pfkB, and nark2), whose expression was significantly lower in M. bovis BCG-pMV261. When the strains were cultured in hypoxia, significantly higher culturability was observed in M. bovis BCG-pMV261::rpfC compared to M. bovis BCG-pMV261. This response was accompanied by a higher sigB and sigE expression. In both strains, we observed a higher dosT, devR, fdxA, and fpkB expression in response to hypoxia. Interestingly, except for fdxA, the expression of these genes was lower in M. bovis BCG-pMV261::rpfC. The protein profiles of M. bovis BCG-pMV261::rpfC reflected the maintenance of an active replicative state (similar to that of the strain grown in normoxic conditions). In anoxic cultures, no significant changes were observed in the expression of hypoxia-response genes. These findings suggest that rpfC may have a significant physiological role in inducing the growth of M. bovis BCG-pMV261::rpfC, which results in the delayed activation of genes related to the transition to anaerobic metabolism.