NrnA Is a Linear Dinucleotide Phosphodiesterase with Limited Function in Cyclic Dinucleotide Metabolism in Listeria monocytogenes
NrnA Is a Linear Dinucleotide Phosphodiesterase with Limited Function in Cyclic Dinucleotide Metabolism in Listeria monocytogenes
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DOI：
10.1128/jb.00206-21
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发表时间：
2022-01-01
影响因子：
3.2
通讯作者：
Huynh, TuAnh N.
中科院分区：
文献类型：
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作者：
Gall, Aaron R.;Hsueh, Brian Y.;Huynh, TuAnh N.
单核增生李斯特菌产生c-二- amp和c-二- gmp来介导许多重要的细胞过程，但这两种核苷酸的水平必须受到调节。c-二- amp的积累会减弱毒力并降低应激反应，而c-二- gmp的积累会损害细菌的运动能力。维持c-二- amp和c-二- gmp稳态的一个重要调控机制是将它们分别水解成线性二核苷酸pApA和pGpG，但这些水解产物的转运尚未在单核增生乳杆菌中得到研究。我们发现NrnA是一种独立的DHH-DHHA1磷酸二酯酶，具有广泛的底物范围，但对线性二核苷酸的偏好强于环二核苷酸。尽管NrnA在体外表现出可检测的环二核苷酸水解活性，但即使在缺乏c-二磷酸二酯酶PdeA和PgpH的情况下，NrnA对细菌细胞中环二核苷酸水解活性的影响也可以忽略不计。Delta nrnA突变体具有哺乳动物细胞感染缺陷，可被大肠杆菌完全修复。总之，我们的数据表明，单核增生乳杆菌的NrnA在功能上与Orn同源，其首选的生理底物很可能是线性二核苷酸。此外，我们的研究结果表明，与其他一些c-二- amp和c-二- gmp生产细菌不同，单核增生L不使用其水解产物来调节其磷酸二酯酶，至少在Delta nrnA突变体中pApA和pGpG水平是如此。最后，通过主毒力调控因子PrfA的组成性激活克服了Delta nrnA感染缺陷，这表明累积的线性二核苷酸抑制了PrfA调控的毒力因子的表达、稳定性或功能。单核增生李斯特菌产生c-二- amp和c-二- gmp，并编码特异性磷酸二酯酶，分别将它们降解为pApA和pGpG，但这些产物的代谢尚未在该细菌中表征。我们发现单核增生乳杆菌的NrnA可以降解多种核苷酸。在测试的环状和线性底物中，它对线性二核苷酸pApA， pGpG和pApG表现出强烈的生化和生理偏好。与其他一些细菌不同，这些寡核苷酸似乎不会干扰环二核苷酸的水解。在肉汤培养中，单核细胞增生L细胞可以很好地耐受NrnA的缺失，但会损害其感染哺乳动物细胞的能力。这些发现表明单核增生乳杆菌的环二核苷酸信号和寡核苷酸代谢是分离的。
Listeria monocytogenes produces both c-di-AMP and c-di-GMP to mediate many important cellular processes, but the levels of both nucleotides must be regulated. c-di-AMP accumulation attenuates virulence and diminishes stress response, and c-di-GMP accumulation impairs bacterial motility. An important regulatory mechanism to maintain c-di-AMP and c-di-GMP homeostasis is to hydrolyze them to the linear dinucleotides pApA and pGpG, respectively, but the fates of these hydrolytic products have not been examined in L. monocytogenes. We found that NrnA, a stand-alone DHH-DHHA1 phosphodiesterase, has a broad substrate range but with a strong preference for linear dinucleotides over cyclic dinucleotides. Although NrnA exhibited detectable cyclic dinucleotide hydrolytic activities in vitro, NrnA had negligible effects on their levels in the bacterial cell, even in the absence of the c-di-AMP phosphodiesterases PdeA and PgpH. The Delta nrnA mutant had a mammalian cell infection defect that was fully restored by Escherichia coli Orn. Together, our data indicate that L. monocytogenes NrnA is functionally orthologous to Orn, and its preferred physiological substrates are most likely linear dinucleotides. Furthermore, our findings revealed that, unlike some other c-di-AMP- and c-di-GMP-producing bacteria, L monocytogenes does not employ their hydrolytic products to regulate their phosphodiesterases, at least at the pApA and pGpG levels in the Delta nrnA mutant. Finally, the Delta nrnA infection defect was overcome by constitutive activation of PrfA, the master virulence regulator, suggesting that accumulated linear dinucleotides inhibit the expression, stability, or function of PrfA-regulated virulence factors.IMPORTANCE Listeria monocytogenes produces both c-di-AMP and c-di-GMP and encodes specific phosphodiesterases that degrade them into pApA and pGpG, respectively, but the metabolism of these products has not been characterized in this bacterium. We found that L. monocytogenes NrnA degrades a broad range of nucleotides. Among the tested cyclic and linear substrates, it exhibits a strong biochemical and physiological preference for the linear dinucleotides pApA, pGpG, and pApG. Unlike in some other bacteria, these oligoribonucleotides do not appear to interfere with cyclic dinucleotide hydrolysis. The absence of NrnA is well tolerated by L monocytogenes in broth cultures but impairs its ability to infect mammalian cells. These findings indicate a separation of cyclic dinucleotide signaling and oligoribonucleotide metabolism in L. monocytogenes.