Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids

Abstract

One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc2155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.

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

“…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). Accordingly, we found a decrease in the cAMP concentration in Mtb cells after 4.5 months of storage which accompanied the significant developing of "non culturability" (Table 1).…”

Free Trehalose Accumulation in Dormant Mycobacterium smegmatis Cells and Its Breakdown in Early Resuscitation Phase

“…Dormant M. smegmatis cells are able to resume their multiplication in fresh medium after some period of ‘metabolic resuscitation’ which precedes cell division (Shleeva et al, 2013). …”

Detection and Quantification of Differentially Culturable Tubercle Bacteria in Sputum from Patients with Tuberculosis

“…This suggests that growth stimulation of organisms in sputum is mediated by both enzymatic and nonenzymatic effects. In this regard, it has been demonstrated that cyclic AMP and fatty acids retain the capacity to modulate the growth of nonreplicating mycobacteria (21)(22)(23).…”