coli encodes only one adenylate cyclase, the adenylate cyclase gene family has undergone a remarkable expansion in mycobacteria.
cAMP then binds to and alters the function of effector proteins like the transcription factor CRP ( Stapleton et al., 2010), the protein lysine acetyltransferase Mt-Pat ( Nambi et al., 2013), and numerous other potential cAMP-binding proteins ( Johnson and McDonough, 2018) to modify Mtb gene expression or gene product activity. Adenylate cyclases sense extracellular or intracellular signals, either directly or indirectly, and transduce this signal into a cellular response by converting ATP into the small molecule second messenger 3’,5’-cyclic-AMP (cAMP) and pyrophosphate ( Johnson and McDonough, 2018). In addition to two-component systems and serine/threonine kinases, one of the most ubiquitous signal transduction modalities in Mtb is the adenylate cyclases. While several examples have been described, it is clear that numerous poorly understood signaling mechanisms exist that promote Mtb survival while reducing the effectiveness of antibiotic therapy ( Bellerose et al., 2020). Thus, the signaling and regulatory networks that facilitate Mtb survival under diverse physiologic conditions can also secondarily reduce the effectiveness of antibiotics. Dephosphorylation reduces CwlM interaction with and activation of the peptidoglycan biosynthetic enzyme MurA, thereby reducing cell wall metabolism and promoting tolerance to starvation and antibiotics. In a second example, nutrient starvation results in the dephosphorylation of CwlM, a substrate of the eukaryotic-like protein serine/threonine kinase PknB ( Boutte et al., 2016). DosR induces a 48 gene regulon which ultimately slows growth, promoting survival under hypoxic conditions and tolerance to antibiotics that are more active against rapidly replicating bacteria ( Galagan et al., 2013). For example, hypoxia reduces Mtb respiratory capacity and activates the DosRST two-component system ( Park et al., 2003). The physiology that enables Mtb to adapt to and persist in the host can also decrease the effectiveness of antibiotics ( Bellerose et al., 2020 Larrouy-Maumus et al., 2016). Mycobacterium tuberculosis (Mtb) has evolved complex signaling and regulatory networks to sense and adapt to the diverse niches through which it transits during infection ( Johnson and McDonough, 2018 Parish, 2014 Richard-Greenblatt and Av-Gay, 2017). Our work defines rv3645 and cAMP as central mediators of intrinsic multidrug resistance and fatty acid metabolism in Mtb and highlights the potential utility of small molecule modulators of cAMP signaling. Using mass spectrometry, we found that Rv3645 is the dominant source of cAMP under standard laboratory growth conditions, that cAMP production is the essential function of Rv3645 in the presence of long-chain fatty acids, and that reduced cAMP levels result in increased long-chain fatty acid uptake and metabolism and increased antibiotic susceptibility. A suppressor screen further identified mutations in the atypical cAMP phosphodiesterase rv1339 that suppress both fatty acid and drug sensitivity phenotypes in strains lacking rv3645. We made the unexpected observation that rv3645 is conditionally essential for Mtb growth only in the presence of long-chain fatty acids, a host-relevant carbon source. We found that a lack of rv3645 resulted in increased sensitivity to numerous antibiotics by a mechanism independent of substantial increases in envelope permeability. Here, we took a genetic approach to investigate the function of the sole essential adenylate cyclase in Mtb H37Rv, Rv3645. Despite this fact, our understanding of how cAMP regulates Mtb physiology remains limited. Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, devotes a considerable amount of coding capacity to produce, sense, and degrade cAMP. Cyclic AMP (cAMP) is a ubiquitous second messenger that transduces signals from cellular receptors to downstream effectors.
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