Dissecting bacterial cell wall synthesis using in vivo single molecule tracking

使用体内单分子追踪剖析细菌细胞壁的合成

• 批准号: 8754253
• 负责人: Ethan Clark Garner
• 金额: $ 227.07万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8754253
• 关键词: Antibiotics; Bacteria; Biochemical; Cell Communication; Cell Shape; Cell Wall; Cells; Communication; Encapsulated; Environment; Enzymes; Genes; Genetic; Growth; Locales; Location; Maps; Motion; Pathway interactions; Peptidoglycan; Polysaccharides; Positioning Attribute; Protein Biosynthesis; Protein Dynamics; Proteins; Reaction; Reading; Regulation; Resistance; Shapes; Stress; Structure; System; Testing; Time; Work; crosslink; environmental change; in vivo; protein protein interaction; public health relevance; retinal rods; single molecule; synthetic enzyme

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.

描述（由申请人提供）：我们不了解细菌生长的机制。杆状细菌通过将新的聚糖链插入细胞壁而拉长。这些链被交联成一个三维网状结构，叫做肽聚糖。这种封装结构不仅定义了细菌的形状，还保护它们不受环境的影响。细胞调节这种结构如何合成、交联或硬化，以适应环境变化、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起作用的：所有合成酶的紧急作用是如何产生均匀生长、形状完美的细胞的。这就要求每个酶的活性在空间中受到调节，使其活性定位在适当的位置。我们也不知道细菌如何及时调节酶的活性：它们如何调节特定的活动来应对外部压力。临床上阻止细胞壁合成的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，通过寻找细胞壁上的其他点

项目成果

The Ribbon-Helix-Helix Domain Protein CdrS Regulates the Tubulin Homolog ftsZ2 To Control Cell Division in Archaea

• DOI: 10.1128/mbio.01007-20
• 发表时间: 2020-07-01
• 期刊: MBIO
• 影响因子: 6.4
• 作者: Darnell, Cynthia L.;Zheng, Jenny;Schmid, Amy K.
• 通讯作者: Schmid, Amy K.

Mechanics and dynamics of translocating MreB filaments on curved membranes

MreB 丝在弯曲膜上易位的力学和动力学

• DOI: 10.7554/elife.40472
• 发表时间: 2019
• 期刊: eLife
• 影响因子: 7.7
• 作者: Wong, Felix;Garner, Ethan C;Amir, Ariel
• 通讯作者: Amir, Ariel

Division plane placement in pleomorphic archaea is dynamically coupled to cell shape.

多形性古细菌中的划分平面位置与细胞形状动态耦合。

• DOI: 10.1111/mmi.14316
• 发表时间: 2019
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Walsh,JamesC;Angstmann,ChristopherN;Bisson-Filho,AlexandreW;Garner,EthanC;Duggin,IainG;Curmi,PaulMG
• 通讯作者: Curmi,PaulMG

The role of cell-envelope synthesis for envelope growth and cytoplasmic density in Bacillus subtilis.

• DOI: 10.1093/pnasnexus/pgac134
• 发表时间: 2022-09
• 期刊: PNAS NEXUS
• 作者: Kitahara, Yuki;Oldewurtel, Enno R.;Wilson, Sean;Sun, Yingjie;Altabe, Silvia;de Mendoza, Diego;Garner, Ethan C.;van Teeffelen, Sven
• 通讯作者: van Teeffelen, Sven

An exhaustive multiple knockout approach to understanding cell wall hydrolase function in Bacillus subtilis.

• DOI: 10.1128/mbio.01760-23
• 发表时间: 2023-10-31
• 期刊: MBIO
• 影响因子: 6.4
• 作者: Wilson, Sean A.;Tank, Raveen K. J.;Hobbs, Jamie K.;Foster, Simon J.;Garner, Ethan C.
• 通讯作者: Garner, Ethan C.