Abstract
Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo.
Highlights
Asymptomatic forms of tuberculosis (TB), when infected individuals carry the pathogen in dormant state without notable symptoms of the disease for a very long time, are termed latent tuberculosis infection (LTBI)
We present a few lines of evidence indicating the importance of adenylyl cyclase (AC) expression and cAMP production for maintaining M. tuberculosis (Mtb) vitality under unfavorable for growth conditions
There was a significant difference in in vitro growth dynamics between the AC-overexpressing pMindRv2212 and the control strain if the development of bacterial population started from a small amount of ancestors (Figure 1)
Summary
Asymptomatic forms of tuberculosis (TB), when infected individuals carry the pathogen in dormant state without notable symptoms of the disease for a very long time, are termed latent tuberculosis infection (LTBI). The role of the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene has been elucidated through a model of dormant Mycobacterium smegmatis resuscitation. M. smegmatis and M. tuberculosis transformation with the plasmid containing the MSMEG_4279 gene expressed under the Tet-promoter, which led to its hyper-expression and an increase in intracellular cAMP concentration, prevented the transition of the two species of bacteria to dormancy under stressful conditions (Shleeva et al, 2013). This suggests that in the presence of high amounts of cAMP provided by the AC the bacteria retained their active state. Direct evidence for the “anti-dormant” role of the M. tuberculosis gene(s) encoding AC for Mtb remained lacking
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