Stress Suppressor Screening Leads to Detection of Regulation of Cyclic di-AMP Homeostasis by a Trk Family Effector Protein in Streptococcus pneumoniae
Stress Suppressor Screening Leads to Detection of Regulation of Cyclic di-AMP Homeostasis by a Trk Family Effector Protein in Streptococcus pneumoniae
复制标题
DOI：
10.1128/jb.00045-18
复制
发表时间：
2018-02
影响因子：
3.2
通讯作者：
Tiffany M. Zarrella;D. Metzger;G. Bai
中科院分区：
文献类型：
--
作者：
Tiffany M. Zarrella;D. Metzger;G. Bai
摘要 环二-AMP（c-di-AMP）是一种新发现的细菌第二信使。然而，人们对 c-di-AMP 稳态的调节知之甚少。在肺炎链球菌中，唯一的二腺苷酸环化酶 CdaA 产生 c-di-AMP，而两种磷酸二酯酶 Pde1 和 Pde2 则裂解信号二核苷酸。为了扩展我们对肺炎球菌 c-di-AMP 信号网络的了解，我们对 Δpde1 Δpde2 热休克抑制剂进行了全基因组测序。除了对热休克生存的影响外，这些抑制突变还恢复了一般的应激抵抗力并改善了在丰富培养基中的生长。 CdaA 或钾转运蛋白 TrkH 中的突变与导致 CdaA C 末端移码的插入配对显着降低了 c-di-AMP 水平。这些观察结果表明，Δpde1 Δpde2 突变体中升高的 c-di-AMP 水平增强了肺炎链球菌对应激条件的敏感性。有趣的是，我们之前已经证明 TrkH 与 Trk 家族 c-di-AMP 结合蛋白 CabP 复合，以介导钾的吸收。在这项研究中，我们发现 cabP 的缺失显着降低了肺炎球菌 c-di-AMP 水平。这是首次观察到 c-di-AMP 效应蛋白调节细菌 c-di-AMP 稳态。重要性 第二信使，包括 c-di-AMP，在细菌物种中普遍存在。在肺炎链球菌中，c-di-AMP磷酸二酯酶编码基因无效突变体在小鼠感染模型中减弱，但c-di-AMP信号在肺炎球菌发病机制中的作用是神秘的。在这项工作中，我们发现热休克抑制突变通过改变细胞内 c-di-AMP 浓度来削弱 c-di-AMP 毒性。这些突变改善了 c-di-AMP 磷酸二酯酶缺陷型肺炎球菌的生长并恢复了应激反应，从而证明了严格调节 c-di-AMP 稳态以应对应激的必要性。同样，这项工作表明 c-di-AMP 效应蛋白 CabP 影响 c-di-AMP 稳态，这为 c-di-AMP 调节提供了新的认识。这项研究对产生 c-di-AMP 的细菌具有重要意义，因为许多物种都含有 CabP 同源物。
ABSTRACT Cyclic di-AMP (c-di-AMP) is a newly discovered bacterial second messenger. However, regulation of c-di-AMP homeostasis is poorly understood. In Streptococcus pneumoniae, a sole diadenylate cyclase, CdaA, produces c-di-AMP and two phosphodiesterases, Pde1 and Pde2, cleave the signaling dinucleotide. To expand our knowledge of the pneumococcal c-di-AMP signaling network, we performed whole-genome sequencing of Δpde1 Δpde2 heat shock suppressors. In addition to their effects on surviving heat shock, these suppressor mutations restored general stress resistance and improved growth in rich medium. Mutations in CdaA or in the potassium transporter TrkH paired with an insertion leading to a frameshift at the C terminus of CdaA significantly reduced c-di-AMP levels. These observations indicate that the elevated c-di-AMP levels in the Δpde1 Δpde2 mutant enhance susceptibility of S. pneumoniae to the stress conditions. Interestingly, we have previously shown that TrkH complexes with a Trk family c-di-AMP-binding protein, CabP, to mediate potassium uptake. In this study, we found that deletion of cabP significantly reduced pneumococcal c-di-AMP levels. This is the first observation that a c-di-AMP effector protein modulates bacterial c-di-AMP homeostasis. IMPORTANCE Second messengers, including c-di-AMP, are prevalent among bacterial species. In S. pneumoniae, c-di-AMP phosphodiesterase-encoding gene null mutants are attenuated during mouse models of infection, but the role of c-di-AMP signaling in pneumococcal pathogenesis is enigmatic. In this work, we found that heat shock suppressor mutations converge on undermining c-di-AMP toxicity by changing intracellular c-di-AMP concentrations. These mutations improve the growth and restore the stress response generally in c-di-AMP phosphodiesterase-deficient pneumococci, thereby demonstrating the essentiality for tight regulation of c-di-AMP homeostasis in order to respond to stress. Likewise, this work demonstrates that a c-di-AMP effector protein, CabP, affects c-di-AMP homeostasis, which provides new perception into c-di-AMP regulation. This study has implications for c-di-AMP-producing bacteria since many species contain CabP homologs.