Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.
Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.
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DOI：
10.1128/mbio.01047-17
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发表时间：
2017-07-25
期刊：
影响因子：
6.4
通讯作者：
Oliver SG
中科院分区：
文献类型：
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作者：
Hesketh A;Vergnano M;Wan C;Oliver SG
我们已经将酿酒酵母工程化以诱导合成原核信号传导核苷酸环二GMP（cdiGMP）、cdiAMP和ppGpp，以表征这些核苷酸在细菌发病过程中对真核细胞功能施加的影响范围。合成遗传阵列（SGA）和转录组分析表明，虽然这些化合物在酵母中引起一些常见的反应，但对三种核苷酸中的每一种也有复杂而独特的反应。所有三种都能够抑制真核细胞生长，其中鸟嘌呤核苷酸表现出比cdiAMP更强的作用。损害线粒体功能和染色质重塑的突变显示与所有三种核苷酸的负上位相互作用。相比之下，某些突变，导致缺陷的染色质修饰和核糖体蛋白功能显示积极上位性，减轻生长抑制的三个核苷酸中的至少两个。独特的是，cdiGMP对通过乙酸呼吸生长的细胞和专性发酵娇小突变体都是致命的。与核糖核苷酸还原酶（RNR）抑制剂羟基脲一起，cdiGMP也是合成致死的。人ppGpp水解酶Mesh1p的异源表达阻止了ppGpp在工程酵母中的积累并恢复了细胞生长。细菌信号分子和真核基因功能之间存在广泛的体内相互作用，导致从生长抑制到死亡的结果。cdiGMP通过必须由不受阻碍的RNR活性或由功能胜任的线粒体补偿的机制发挥作用。可能需要Mesh1p来消除ppGpp在受到细菌感染的人类细胞中的破坏作用。在感染过程中，病原菌可以将核苷酸释放到其真核宿主的细胞中。这些核苷酸被认为是有助于启动防御性免疫反应的信号，帮助受感染的细胞恢复。尽管这一过程的重要性，细菌核苷酸对真核细胞功能的更广泛的影响仍然不清楚。为了解决这个问题，我们对真核生物酿酒酵母（面包酵母）的细胞进行了遗传修饰，以产生其中三种分子（cdiAMP，cdiGMP和ppGpp），并使用工程菌株作为模型系统来表征分子对细胞的影响。除了证明核苷酸各自能够对酵母细胞功能和生长产生不利影响外，我们还确定了对减轻所造成的损害重要的细胞功能，从而提出了可能的作用模式。这项研究扩展了我们对细菌和真核细胞之间可能发生的分子相互作用的理解。
We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected cells recover. Despite the importance of this process, the broader impact of bacterial nucleotides on the functioning of eukaryotic cells remains poorly defined. To address this, we genetically modified cells of the eukaryote Saccharomyces cerevisiae (baker’s yeast) to produce three of these molecules (cdiAMP, cdiGMP, and ppGpp) and used the engineered strains as model systems to characterize the effects of the molecules on the cells. In addition to demonstrating that the nucleotides are each capable of adversely affecting yeast cell function and growth, we also identified the cellular functions important for mitigating the damage caused, suggesting possible modes of action. This study expands our understanding of the molecular interactions that can take place between bacterial and eukaryotic cells.