Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice

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.

One-Year Old Dormant, “Non-culturable” Mycobacterium tuberculosis Preserves Significantly Diverse Protein Profile

“…We also checked the intracellular concentration of cAMP. Previously we found that the transition of Mtb to the dormant "non-culturable" state correlates with a decrease in the cAMP concentration (Shleeva et al, 2017a). Alternatively, cAMP concentration increases in Msm cells under resuscitation (Shleeva et al, 2013).…”

Mycobacterial Control of Host Mitochondria: Bioenergetic and Metabolic Changes Shaping Cell Fate and Infection Outcome

“…. Other adenylate cyclases influencing mycobacterial virulence include Rv2212 (Shleeva et al, 2017) and Rv1675c (Smith et al, 2017). Unlike cAMP, c-di-AMP, and c-di-GMP serve as an alarm signal for the mammalian immune system (Karaolis et al, 2007).…”

Prolonged infection triggered by dormant Mycobacterium tuberculosis: Immune and inflammatory responses in lungs of genetically susceptible and resistant mice

“…Assuming that as longer is the duration of experimental infection, the higher is precision of its dynamic assessment and relevance to the LTBI problem, we began to use M. tuberculosis strains with substantially diminished virulence, which induce very slowly developing disease in mice. Two main approaches of M. tuberculosis H37Rv attenuation were applied: genetic attenuation caused by knocking out genes of the Rpf family involved in regulation of mycobacterial growth [21][22][23], and a prolonged exposure to a gradual acidification in vitro, resulting in a so-called "non-culturable", dormant-like state of mycobacteria [24,25]. Using the first approach, we revealed previously unknown features of early and late lung inflammation that differentiate genetically susceptible and resistant mice.…”