Cyclic di-AMP is critical for Listeria monocytogenes growth, cell wall homeostasis, and establishment of infection.
Cyclic di-AMP is critical for Listeria monocytogenes growth, cell wall homeostasis, and establishment of infection.
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DOI：
10.1128/mbio.00282-13
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发表时间：
2013-05-28
期刊：
影响因子：
6.4
通讯作者：
Woodward JJ
中科院分区：
文献类型：
--
作者：
Witte CE;Whiteley AT;Burke TP;Sauer JD;Portnoy DA;Woodward JJ
单核细胞增生李斯特菌感染导致被称为胞质监视途径（CSP）的先天免疫信号传导途径的稳健诱导，其特征在于β干扰素（IFN-β）和共调节基因的表达。我们先前将IFN-β刺激配体鉴定为分泌的环状二AMP。单核细胞增生李斯特菌中c-di-AMP的合成由二腺苷酸环化酶DacA催化，并且多药耐药转运蛋白是分泌所必需的。为了鉴定CSP检测单核细胞增生李斯特菌中涉及的其他细菌因素，我们对诱导IFN-β水平改变的突变体进行了正向遗传筛选。一种刺激IFN-β水平升高的突变体在基因lmo 0052中携带转座子插入。Lmo 0052，在此重新命名为PdeA，与枯草芽孢杆菌的环状二AMP磷酸二酯酶GdpP（以前称为YybT）具有同源性，并且能够将c-di-AMP降解为线性二核苷酸pApA。通过二腺苷酸环化酶DacA或PdeA过表达的条件性耗竭减少c-di-AMP水平导致体外和巨噬细胞生长速率显著降低。此外，c-di-AMP水平改变的突变体对肽聚糖靶向抗生素具有不同的敏感性，这表明该分子可能参与调节细胞壁稳态。在细胞内感染期间，c-di-AMP产生的增加导致CSP的过度活化。dacA的条件性消耗也导致IFN-β表达增加和伴随的宿主细胞焦亡增加，这是细菌溶解增加和随后细菌DNA释放的结果。这些数据表明，c-di-AMP协调细菌生长，细胞壁稳定性和对应激的反应，并在细菌感染的建立中起着至关重要的作用。单核细胞增生李斯特菌是一种革兰氏阳性的胞内病原菌，是食源性疾病李斯特菌病的病原体。感染后，单核细胞增生李斯特菌刺激IFN-β和共调节基因的表达，这取决于宿主检测分泌的细菌信号传导核苷酸c-di-AMP。使用诱导宿主IFN-β高水平表达的突变体的正向遗传筛选，我们鉴定了降解c-di-AMP的c-di-AMP磷酸二酯酶PdeA。在这里，我们表征单核细胞增多性李斯特菌突变体表达增强或减少水平的c-di-AMP。通过二腺苷酸环化酶（DacA）的条件性耗竭或PdeA的过表达降低c-di-AMP水平减弱细菌生长并导致细菌溶解，这表明其产生对于生存力是必不可少的，并且可以调节细胞壁代谢。缺乏PdeA的突变体具有独特的转录谱，这可能为该分子的其他作用提供见解。这项工作表明，c-di-AMP是细菌复制，细胞壁稳定性和致病性所需的关键信号分子。
Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. Listeria monocytogenes is a Gram-positive intracellular pathogen and the causative agent of the food-borne illness listeriosis. Upon infection, L. monocytogenes stimulates expression of IFN-β and coregulated genes dependent upon host detection of a secreted bacterial signaling nucleotide, c-di-AMP. Using a forward genetic screen for mutants that induced high levels of host IFN-β expression, we identified a c-di-AMP phosphodiesterase, PdeA, that degrades c-di-AMP. Here we characterize L. monocytogenes mutants that express enhanced or diminished levels of c-di-AMP. Decreased c-di-AMP levels by conditional depletion of the diadenylate cyclase (DacA) or overexpression of PdeA attenuated bacterial growth and led to bacteriolysis, suggesting that its production is essential for viability and may regulate cell wall metabolism. Mutants lacking PdeA had a distinct transcriptional profile, which may provide insight into additional roles for the molecule. This work demonstrates that c-di-AMP is a critical signaling molecule required for bacterial replication, cell wall stability, and pathogenicity.