cAMP and antimicrobial susceptibility in mycobacteriaAntimicrobial tolerance, the ability to survive exposure to antimicrobials via transient nonspecific means, promotes the development of antimicrobial resistance (AMR). The study of the molecular mechanisms that result in antimicrobial tolerance is therefore essential for the understanding of AMR. In gram-negative bacteria, the second messenger molecule 3′’,5′’-cAMP has been previously shown to be involved in AMR. In mycobacteria, however, the role of cAMP in antimicrobial tolerance has been difficult to probe due to its particular complexity. In order to address this difficulty, here, through unbiased biochemical approaches consisting in the fractionation of clear protein lysate from a mycobacterial strain deleted for the known cAMP phosphodiesterase (Rv0805c) combined with mass spectrometry techniques, we identified a novel cyclic nucleotide-degrading phosphodiesterase enzyme (Rv1339) and developed a system to significantly decrease intracellular cAMP levels through plasmid expression of Rv1339 using the constitutive expression system, pVV16. In Mycobacterium smegmatis mc²155, we demonstrate that recombinant expression of Rv1339 reduced cAMP levels threefold and resulted in altered gene expression, impaired bioenergetics, and a disruption in peptidoglycan biosynthesis leading to decreased tolerance to antimicrobials that target cell wall synthesis such as ethambutol, D-cycloserine, and vancomycin. This work increases our understanding of the role of cAMP in mycobacterial antimicrobial tolerance, and our observations suggest that nucleotide signaling may represent a new target for the development of antimicrobial therapies.

分枝杆菌中的 cAMP 和抗菌素敏感性抗菌素耐受性，暴露于抗菌素后存活的能力短暂的非特异性手段，促进抗菌素耐药性 (AMR) 的发展。因此，研究导致抗菌素耐受的分子机制对于理解 AMR 至关重要。在革兰氏阴性菌中，第二信使分子 3'',5''-cAMP 先前已被证明与 AMR 有关。然而，在分枝杆菌中，由于其特殊的复杂性，cAMP 在抗菌素耐受性中的作用一直难以探究。为了解决这一难题，在此，通过无偏生化方法，包括从已知 cAMP 磷酸二酯酶 (Rv0805c) 缺失的分枝杆菌菌株中分离出透明蛋白裂解物，并结合质谱技术，我们鉴定了一种新型环核苷酸降解磷酸二酯酶 (Rv1339)，并开发了一个系统，通过使用组成型表达系统 pVV16 的 Rv1339 质粒表达显着降低细胞内 cAMP 水平。在耻垢分枝杆菌mc ² 155，我们证明 Rv1339 的重组表达可将 cAMP 水平降低三倍，并导致基因表达改变、生物能量学受损以及肽聚糖生物合成中断，从而导致对靶向细胞壁合成的抗菌剂（例如乙胺丁醇、D-环丝氨酸和万古霉素。这项工作增加了我们对 cAMP 在分枝杆菌抗菌耐受性中的作用的理解，我们的观察表明核苷酸信号可能代表了抗菌疗法开发的新目标。