Chemical activation of adenylyl cyclase Rv1625c inhibits growth of Mycobacterium tuberculosis on cholesterol and modulates intramacrophage signaling

Abstract

Mycobacterium tuberculosis (Mtb) uses a complex 3′-5′-cyclic AMP (cAMP) signaling network to sense and respond to changing environments encountered during infection, so perturbation of cAMP signaling might be leveraged to disrupt Mtb pathogenesis. However, understanding of cAMP signaling pathways is hindered by the presence of at least 15 distinct adenylyl cyclases (ACs). Recently, the small molecule V-58 was shown to inhibit Mtb replication within macrophages and stimulate cAMP production in Mtb. Here we determined that V-58 rapidly and directly activates Mtb AC Rv1625c to produce high levels of cAMP regardless of the bacterial environment or growth medium. Metabolic inhibition by V-58 was carbon source dependent in Mtb and did not occur in Mycobacterium smegmatis, suggesting that V-58-mediated growth inhibition is due to interference with specific Mtb metabolic pathways rather than a generalized cAMP toxicity. Chemical stimulation of cAMP production by Mtb within macrophages also caused down regulation of TNF-α production by the macrophages, indicating a complex role for cAMP in Mtb pathogenesis. Together these studies describe a novel approach for targeted stimulation of cAMP production in Mtb, and provide new insights into the myriad roles of cAMP signaling in Mtb, particularly during Mtb's interactions with macrophages.

Cyclic nucleotide signaling in Mycobacterium tuberculosis: an expanding repertoire

“…tissue culture media alone (Bai, Schaak and McDonough 2009). Mtb also elevates the production and secretion of cAMP when propionate is present in the medium (Johnson et al 2017), which is consistent with reports of cAMP-dependent regulation of enzymes involved in propionate metabolism (Nambi, Basu and Visweswariah 2010;Xu, Hegde and Blanchard 2011;Hayden et al 2013;Nambi et al 2013).…”

“…As previously mentioned, Mtb grown on propionate produces and secretes over 10 times the amount of cAMP found in Mtb grown on glycerol as the primary carbon source (Johnson et al 2017). cAMP produced by Mtb in the presence of propionate or its metabolites may control the accumulation of propionyl-coA.…”

“…Another exciting recent development is the discovery of a small molecule inhibitor of Mtb, V-58, which directly and specifically activates AC Rv1625c (VanderVen et al 2015;Johnson et al 2017). Rv1625c functions as a membrane-associated homodimer in which each Rv1625c monomer is expected to contribute six TM domains and one catalytic site to resemble the characteristic architecture of mammalian tmACs (Guo et al 2001).…”

“…V-58 is proposed to fit into one of two putative active sites in the Rv1625c dimer, in a manner similar to that of forskolin (Johnson et al 2017). Forskolin is used routinely to study the impact of elevated cAMP on various aspects of cell physiology, and its discovery revolutionized the field of cAMP signaling in eukaryotic organisms (reviewed by Alasbahi and Melzig 2012).…”

AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non‐coding small RNA Mcr11

“…The universal second messenger molecule 3′, 5′ cyclic adenosine monophosphate (cAMP) is a global regulator of gene expression within Mtb, while secreted cAMP modulates Mtb's interactions with host macrophages (Lowrie et al, 1979;Agarwal et al, 2009;Bai et al, 2009;Nambi et al, 2013;Kahramanoglou et al, 2014;Knapp et al, 2015;Ranganathan et al, 2016;Johnson et al, 2017). cAMP levels affect the gene regulatory activities of two virulence-associated CRP/FNR family transcription factors in Mtb, Cmr and CRP Mt (Bai et al, 2005;Rickman et al, 2005;Kahramanoglou et al, 2014;Ranganathan et al, 2016).…”