Dissecting bacterial cell wall synthesis using in vivo single molecule tracking

描述（由申请人提供）：我们不了解细菌生长的机制。杆状细菌通过在细胞壁中插入新的聚糖链来伸长。这些链被交联成一个称为肽聚糖的三维网络。这种封装结构不仅定义了细菌的形状，还保护它们免受环境的影响。细胞调节这种结构的合成、交联或硬化方式，以便它们能够适应环境变化、外部压力或抗生素的存在。细菌使用许多不同的酶来构建和修饰细胞壁，虽然我们知道每种酶催化的反应，但我们不知道这些酶在体内是如何发挥作用的：所有合成酶的紧急作用如何创造均匀生长的完美形状的细胞。这就要求每种酶的活性在空间上受到调节，使其活性定位在适当的位置。也不知道细菌如何及时调节酶活性：它们如何调节特定的活性以应对外部压力。阻止细胞壁合成的临床抗生素针对的是构建细胞壁的酶。这些抗生素多年来一直有效，但对这类抗生素的耐药性日益蔓延，限制了它们的效用。因此，通过在细胞壁中找到其他点 合成途径，脆弱的点，我们可能会发现新的蛋白质，我们可以靶向抑制细菌生长。而不是酶本身，我们将寻求针对细胞用来调节合成酶活性的机制。据信，这种调节是通过与这些酶相关的蛋白质发生的，改变了它们的位置或活性。遗传和生物化学研究产生了大量可能的调节因子，但是， 互动地图仍不清楚：我们不知道这个系统中的哪些成分相互作用，这些相互作用如何调节细胞壁的合成，更不知道这种相互作用在细胞中何时何地发生。我们已经发现，我们可以读出的协会和活动的细胞壁合成蛋白质的定量运动使用体内单分子跟踪。我们将使用这种方法来剖析细胞壁合成，绘制出蛋白质-蛋白质相互作用以及不同酶功能之间发生的通信。首先，我们通过描述所有已知组件的动态来构建交互图。然后，我们将使用遗传扰动来测试这张地图。为了了解信息是如何在这个网络中传递的，我们将在系统地消耗其他基因时观察每个蛋白质的动态。最后，我们将通过研究天然调节点来确定这个系统中的脆弱点：细菌如何控制它们的生长速度，以及它们如何对抗生素应激作出反应。

DESCRIPTION (provided by applicant): We do not understand the mechanisms that bacteria use to grow. Rod shaped bacteria elongate by inserting new strands of glycans into their cell wall. These strands are cross-linked into a three-dimensional meshwork called peptidoglycan. This encapsulating structure not only defines the bacterial shape, it also protects them from the environment. Cells modulate how this structure is synthesized, cross-linked, or stiffened so that they can adapt to environmental changes, external stresses, or the presence of antibiotics. There are many different enzymes bacteria use to build and modify the cell wall, and while we know the reactions catalyzed by each enzyme, we do not know how these enzymes function in vivo: how does the emergent action of all synthetic enzymes create uniformly growing, perfectly shaped cells. This requires that the activity of each enzyme to be regulated in space so its activity is positioned at the proper locale. It is also not known how bacteria regulate enzymatic activity in time: how they modulate specific activities to respond to external stresses. The clinicl antibiotics that stop cell wall synthesis target the enzymes that build the cell wall. These antibiotics have been effective for many years, but the increasing spread of resistance to this class of antibiotics is limiting their utility. Therefore, by finding other points in the cell wall synthesis pathway, points that are fragile, we may find new proteins we can target to inhibit bacterial growth. Rather than the enzymes themselves, we will seek to target the mechanisms the cell uses to regulate the activity of the synthetic enzymes. It is believed this regulation occurs through proteins that associate with these enzymes, changing their location or activity. Genetic and biochemical studies are yielding a large list of possible regulating factors, but, this interaction map remains unclear: we do not know which components in this system interact, how these interactions modulate cell wall synthesis, much less when or where in the cell this interaction occurs. We have discovered that we can read out the associations and activity of cell wall synthesis proteins by quantitating their motions using in vivo single molecule tracking. We will use this approach to dissect cell wall synthesis, mapping out the protein-protein interactions as well as the communication that occurs between different enzymatic functions. First we build an interaction map by characterizing the dynamics of all known components. We then will test this map using genetic perturbations. To understand how information is communicated within this network, we will observe each proteins dynamics as we systematically deplete other genes. Finally, we will determine what the fragile points are in this system by studying native points of regulation: how bacteria control their rate of growth, and how they respond to antibiotic stresses.