Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction between a Diguanylate Cyclase and Its Receptor.
Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction between a Diguanylate Cyclase and Its Receptor.
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DOI：
10.1128/mbio.01254-18
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发表时间：
2018-07-10
期刊：
影响因子：
6.4
通讯作者：
O'Toole GA
中科院分区：
文献类型：
--
作者：
Giacalone D;Smith TJ;Collins AJ;Sondermann H;Koziol LJ;O'Toole GA
细菌细胞内第二信使，环二聚GMP（c-di-GMP），调节许多细菌的生物膜形成。c-di-GMP通过内膜蛋白LapD的结合控制生物膜形成，并且LapD受体是c-di-GMP介导的生物膜形成的复杂网络的中心。在这项研究中，我们研究了如何c-di-GMP信号特异性的二鸟苷酸环化酶（DGC），GcbC，是通过与LapD受体的相互作用，并通过小配体传感通过GcbC的钙通道趋化性（CACHE）域实现。我们提供的证据表明，生物膜的形成刺激环境相关的有机酸柠檬酸盐（和相关化合物，异柠檬酸盐）在GcbC依赖的方式通过增强GcbC-LapD相互作用，这导致在增加的LapA定位到细胞表面。此外，GcbC在分离时几乎没有合成c-di-GMP的能力。然而，当LapD存在时，GcbC活性显著增强（~8倍），表明与LapD受体结合刺激了该DGC的活性;柠檬酸盐增强的GcbC-LapD相互作用进一步刺激了c-di-GMP合成。我们建议，I-网站的GcbC服务两个角色以外的变构控制这种酶：促进GcbC-LapD相互作用和稳定的GcbC-LapD复合物中的GcbC的活性构象。最后，考虑到LapD可以与荧光假单胞菌的十几种不同的DGC相互作用，其中许多具有配体结合结构域，这里描述的通过LapD-GcbC相互作用的配体介导的增强信号传导可能是该网络中信号传导的保守机制。与这个想法一致，我们确定了第二个例子，配体介导的增强DGC-LapD相互作用，促进生物膜形成。在许多细菌中，数十种酶产生二核苷酸信号c-di-GMP;然而，目前尚不清楚在这种可溶性信号的背景下如何减轻不希望的串扰，以及c-di-GMP信号传导如何受到环境输入的调节。我们证明，GcbC，DGC，显示几乎没有能力合成c-di-GMP在其同源受体LapD的情况下，GcbC-LapD相互作用增强C-di-GMP合成GcbC，可能介导的I-站点的GcbC。我们进一步证明了配体介导的信号传导特异性机制，通过增加DGC与其同源受体的物理相互作用。我们设想了一种情况，其中弱活性DGC的“云”可以通过响应于适当的环境信号与其受体的特异性相互作用来增加其活性，同时促进c-di-GMP产生、配体特异性信号传导和生物膜形成。
The bacterial intracellular second messenger, cyclic dimeric GMP (c-di-GMP), regulates biofilm formation for many bacteria. The binding of c-di-GMP by the inner membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbC’s calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present, GcbC activity is significantly enhanced (~8-fold), indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the I-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of Pseudomonas fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation. In many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP; however, it is unclear how undesired cross talk is mitigated in the context of this soluble signal and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-di-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the I-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a “cloud” of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling, and biofilm formation.