Abstract
Under unfavorable conditions such as host immune responses and environmental stresses, human pathogen Mycobacterium tuberculosis may acquire the dormancy phenotype characterized by “non-culturability” and a substantial decrease of metabolic activity and global transcription rates. Here, we found that the transition of M. tuberculosis from the dormant “non-culturable” (NC) cells to fully replicating population in vitro occurred not earlier than 7 days after the start of the resuscitation process, with predominant resuscitation over this time interval evidenced by shortening apparent generation time up to 2.8 h at the beginning of resuscitation. The early resuscitation phase was characterized by constant, albeit low, incorporation of radioactive uracil, indicating de novo transcription immediately after the removal of the stress factor, which resulted in significant changes of the M. tuberculosis transcriptional profile already after the first 24 h of resuscitation. This early response included transcriptional upregulation of genes encoding enzymes of fatty acid synthase system type I (FASI) and type II (FASII) responsible for fatty acid/mycolic acid biosynthesis, and regulatory genes, including whiB6 encoding a redox-sensing transcription factor. The second resuscitation phase took place 4 days after the resuscitation onset, i.e., still before the start of active cell division, and included activation of central metabolism genes encoding NADH dehydrogenases, ATP-synthases, and ribosomal proteins. Our results demonstrate, for the first time, that the resuscitation of dormant NC M. tuberculosis is characterized by immediate activation of de novo transcription followed by the upregulation of genes controlling key metabolic pathways and then, cell multiplication.
Highlights
One-third of the human population is estimated to be latently infected with Mycobacterium tuberculosis, a causative agent of tuberculosis (TB), without overt disease symptoms (World Health Organization, 2018)
Dormancy is a specific physiological state characterized by significant cessation of metabolic activity and growth, whereas resuscitation from dormancy is a process of restoring cell activity followed by bacterial multiplication, which in case of M. tuberculosis can lead to disease progression
There were no changes in the optical density (OD) 600 or most probable number (MPN) corresponding to the maximum count of potentially viably cells in the population until day 7, the number of culturable bacteria estimated by colony forming units (CFU) increased by about 10 times immediately after washing and continued to rise, reaching 5.7 × 107 CFU/ml at day 7 (Figure 1)
Summary
One-third of the human population is estimated to be latently infected with Mycobacterium tuberculosis, a causative agent of tuberculosis (TB), without overt disease symptoms (World Health Organization, 2018). Being under immune control of the host, latent TB infection presents a constant risk of disease reactivation, which constitutes 10% over a lifetime in the general. Understanding the mechanisms underlying M. tuberculosis resuscitation may provide clues to the development of control measures in order to inhibit reactivation of latent infection, reduce disease severity in infected patients, and prevent pathogen transmission in the population. There are several in vivo models of M. tuberculosis latency and reactivation, including mice (McCune and Tompsett, 1956; Scanga et al, 1999; Radaeva et al, 2005), guinea pigs (Ordway et al, 2010), and rabbits (Manabe et al, 2008; Subbian et al, 2012), they do not reproduce disease pathology and immune control observed in humans. The existing in vivo TB reactivation models are aimed to study host immune response after the reactivation of infection rather than the mechanisms of bacterial resuscitation (Mehra et al, 2011; Foreman et al, 2016)
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