Cyclic-AMP Dynamics and Heterogeneity in Mycobacterium tuberculosis: Insights into Host-Pathogen Interactions

Abstract

3′,5′-Cyclic adenosine monophosphate (cAMP) is a nucleotide secondary messenger molecule widely used across biological domains. While it has been implicated in several crucial physiological processes of Mycobacterium tuberculosis, including virulence, resuscitation from dormancy, adaptation to stresses, and antibiotic tolerance, its temporal dynamics and heterogeneity remain unexplored. Using a novel fluorescent biosensor for cAMP, rmgCarvi, we assessed the temporal dynamics of intramycobacterial cAMP during resuscitation from dormancy and dormancy-like states, in response to physiologically relevant stresses in vitro, and during infection in RAW264.7 macrophages. We demonstrate a rapid and large increase in intramycobacterial cAMP levels during resuscitation from nutrient starvation-induced dormancy, independent of the presence of exogenous free fatty acids and reveal new insights into the kinetics of this phenomenon. We also investigated cAMP levels upon infection in RAW264.7 macrophages and confirmed the previously reported increase in intramycobacterial cAMP levels. To delineate the host microenvironment behind this burst, we explored intramycobacterial cAMP dynamics in response to combined acid and salt stress in vitro, generating new insights on the nature of their synergistic regulation. Additionally our studies reveal that acidic pH of the phagosome is not the major driver of increased intramycobacterial cAMP production and revealed tight temporal regulation over the cAMP burst. In conclusion our studies using the cAMP fluorescent reporter strain reveal fundamental insights into general principles of cAMP signalling and regulation in response to stresses and during infection.