Cyclic di-AMP regulation of osmotic homeostasis is essential in Group B Streptococcus.
Cyclic di-AMP regulation of osmotic homeostasis is essential in Group B Streptococcus.
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DOI：
10.1371/journal.pgen.1007342
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发表时间：
2018-04
期刊：
影响因子：
4.5
通讯作者：
Firon A
中科院分区：
文献类型：
--
作者：
Devaux L;Sleiman D;Mazzuoli MV;Gominet M;Lanotte P;Trieu-Cuot P;Kaminski PA;Firon A
环核苷酸普遍用作二级信使来控制细胞生理。在这些信号分子中，环二腺苷酸（c - di - AMP）是一种特定的细菌二级信使，在感染过程中可被宿主细胞识别，并且其合成通过控制一种保守且必需的细胞功能被认为对细菌生长是必需的。在这项研究中，我们试图确定无乳链球菌（新生儿败血症和脑膜炎的病原体）中依赖c - di - AMP的主要途径。通过条件性失活dacA（唯一的二腺苷酸环化酶基因），我们证实c - di - AMP合成在标准生长条件下是必需的。然而，由于补偿性突变的积累，c - di - AMP合成很快变得可有可无。我们鉴定了几个恢复ΔdacA突变体活力的突变，特别是渗透保护剂转运蛋白BusAB的功能缺失突变。对c - di - AMP结合蛋白的鉴定揭示了一组保守的钾离子和渗透调节物质转运蛋白以及BusR转录因子。我们表明BusR通过直接结合busAB启动子负调控busAB转录。如果培养基中存在渗透保护剂（如甘氨酸甜菜碱），在缺乏c - di - AMP的情况下，BusR抑制作用的缺失会导致busAB的毒性表达。相反，gdpP c - di - AMP磷酸二酯酶的缺失会导致高渗敏感性，这是一种依赖于功能性BusR的表型。综上所述，我们证明c - di - AMP对渗透平衡是必需的，并且主要机制依赖于c - di - AMP结合转录因子BusR。渗透平衡的调节可能是c - di - AMP保守且必需的功能，但每个物种都进化出了特定的c - di - AMP渗透调节机制以适应其环境。
基于核苷酸的二级信使在细菌生理和宿主 - 病原体相互作用中起核心作用。在这些信号核苷酸中，环二腺苷酸（c - di - AMP）的合成最初被认为对细菌生长是必需的。在这项研究中，我们证实机会致病菌无乳链球菌中唯一的二腺苷酸环化酶在标准生长条件下是必需的。然而，通过在参与渗透调节的基因中积累自发突变，c - di - AMP合成很快变得可有可无。我们鉴定出c - di - AMP直接与维持渗透平衡所必需的四种蛋白质结合，包括三种渗透调节物质转运蛋白和BusR转录因子。我们证明BusR负调控busAB操纵子的表达，并且如果环境中存在渗透保护剂，它是在缺乏c - di - AMP合成时导致生长抑制的主要成分。总体而言，c - di - AMP通过协调渗透调节物质的摄取对维持渗透平衡是必需的，因此细菌已经发展出特定机制使c - di - AMP成为渗透平衡的核心调节剂。
Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment. Nucleotide-based second messengers play central functions in bacterial physiology and host-pathogen interactions. Among these signalling nucleotides, cyclic-di-AMP (c-di-AMP) synthesis was originally assumed to be essential for bacterial growth. In this study, we confirmed that the only di-adenylate cyclase enzyme in the opportunistic pathogen Streptococcus agalactiae is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable by accumulating spontaneous mutations in genes involved in osmotic regulation. We identified that c-di-AMP binds directly to four proteins necessary to maintain osmotic homeostasis, including three osmolyte transporters and the BusR transcriptional factor. We demonstrated that BusR negatively controls the expression of the busAB operon and that it is the main component leading to growth inhibition in the absence of c-di-AMP synthesis if osmoprotectants are present in the environment. Overall, c-di-AMP is essential to maintain osmotic homeostasis by coordinating osmolyte uptake and thus bacteria have developed specific mechanisms to keep c-di-AMP as the central regulator of osmotic homeostasis.
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