Characterization of new toxins (YmgD and YdfD) from E.coli, targeting cell wall

针对细胞壁的大肠杆菌新毒素（YmgD 和 YdfD）的表征

• 批准号: 8314320
• 负责人: Hisako Masuda
• 金额: $ 5.09万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8314320
• 关键词: Alanine; Amino Acids; Anabolism; Antibiotics; Antitoxins; Apoptosis; Bacteria; Bacterial Genome; Bacterial Physiology; Bacteriophages; Biochemical; Biochemical Reaction; Biogenesis; Biological Assay; Biology; Cell Death; Cell Survival; Cell Wall; Cell division; Cell physiology; Cells; Centrifugation; Chromosomes; Complex; Cytolysis; Cytosol; DNA; Development; Drug Design; Enzymes; Escherichia coli; Genes; Genome; Growth; In Vitro; Investigation; Label; Lead; Life; Lipoproteins; Membrane; Messenger RNA; Methods; Molecular; Monitor; Multi-Drug Resistance; Operon; Organism; Outcome Study; Pathogenicity; Pathway interactions; Penicillin-Binding Proteins; Peptide Signal Sequences; Peptides; Peptidoglycan; Peptidyltransferase; Physiologic pulse; Plasmids; Play; Polymers; Population; Process; Proteins; Reaction; Regulation; Research; Research Project Grants; Ribosomes; Role; System; Targeted Toxins; Testing; Therapeutic; Toxic effect; Toxin; base; cell growth; cellular targeting; computer program; crosslink; in vitro Assay; insight; killings; novel; novel strategies; overexpression; pathogen; pathogenic bacteria; periplasm; prevent; research study; stem; suicidal; theories; transpeptidation

DESCRIPTION (provided by applicant): Bacteria are generally considered to be a single cellular, free living organism, yet they carry toxin or suicidal genes on their genome. These toxins are usually coexpressed with their cognate antitoxins. The sets of these toxin and antitoxin pairs are most often encoded from the single operon, and termed TA systems. The first such example was found in plasmids and bacteriophage, and required for "post-segregational killing' (15, 17). The TA systems are now widely found not only on plasmids and on bacteriophages, but also in the chromosome of almost all bacteria. E. coli carries at least 29 sets of toxin-antitoxin (TA) systems, but the mechanisms of action are deciphered in only a small number of them. Known toxins inhibit a wide range of cellular functions including DNA, mRNA, 30S and 50S ribosome subunits and cell division (17, 30, 37, 38). The exact role of these toxins on bacterial physiology is not yet clearly understood. One possible role is to regulate cell growth under certain severe growth conditions for survival (bacteriostatic). Another theory is that toxins are suicidal, killing unwanted cells in order to maintain a desired population (bacteriocidal), as seen in post-segregational killing (6). The ymgG-ymgD and dicB-ydfD operons from the E. coli chromosome have been identified as possible TA systems using a computer program to predict TA systems (RASTA) in bacterial genomes (27). Both ymgD and ydfD encode for a short peptide (consisting of 109 and 65 amino acid residues, respectively) which are toxic to cell upon expression. However, their cellular targets have not been identified. On the basis of preliminary results from my study, the targets of these toxins appear to be the cell wall, causing a rapid decrease in cell viability upon their induction. This is the first example of TA toxins targeting cell walls to cause cell death. Interestingly, YmgD is produced with a signal peptide and is the first toxin discovered to be secreted into the periplasmic space. In this application, I will attempt to decipher the exact molecular reactions in peptidoglycan (PG) biogenesis, which these toxins target. I will propose a research project examining how YdfD reduces the degree of PG crosslinks. When YdfD expression is induced, 4'-3' crosslinks (between 4' D-alanine residue of one PG stem and 3' meso-DAP of another) are specifically reduced. I predict that YdfD may inhibit the formation of 4'-3' crosslinks, thus weakening the crosslinking of PG polymer causing cell to lyse. In order to unveil the mechanisms of how YdfD reduces the crosslinking, I will first examine the effect of YdfD expression on the specific enzymatic reactions necessary for polymer formation using purified proteins in in vitro assay systems. Chromatographic analysis of precursors will be also performed to examine the accumulation/lack of biosynthetic intermediates. The peptidoglycan purified after the induction of YmgD lacks most of the peptidoglycan crosslinked lipoprotein. Combining this result with our preliminary result that YmgD toxicity was reduced in the lipoprotein deficient strain, we hypothesized that YmgD toxicity is lipoprotein dependent. We have found that YmgD interact with two subunits of the outer membrane lipoprotein translocator, Lol complex. Lol complex is responsible for translocating outer membrane lipoproteins, including Lpp, from inner the membrane to the outer membrane. There are more than 90 outer membrane lipoproteins that are known and serve essential functions. Inhibition of Lol systems is known to cause cell lysis due to the accumulation of lipoproteins in the inner membrane. YmgD may exert its toxicity through inhibition of Lol system, causing improper localization of lipoproteins. I will test this hypothesis through purification of lipoproteins from membranes by differential centrifugation and look for any unusual accumulation of mislocalized lipoproteins. The effect of YmgD on the activity of Lol complex will also be assessed using in vitro system. The research proposed here will advance the understanding of complex bacterial physiology controlled by various TA systems. Since a number of pathogenic bacteria also carry numerous TA systems, and are potentially involved in pathogenicity, further characterization of the TA systems will provide a clue to develop a new strategy for treating pathogens. Also, since the toxins target the cell wall and cause rapid cell lysis, our investigation will directly provide us a new means to examine the basic biology of cell wall biosynthesis, and will reveal a new target of drug design and also lead to develop novel antibiotics.

描述(申请人提供)：细菌通常被认为是一个单一的细胞，自由生活的有机体，但他们携带毒素或自杀基因在他们的基因组。这些毒素通常与它们的同源抗毒素共表达。这些毒素和抗毒素对的集合通常由单个操纵子编码，并被称为TA系统。第一个这样的例子是在质粒和噬菌体中发现的，并且是“分离后杀死”所必需的(15，17)。TA系统现在不仅广泛存在于质粒和噬菌体上，而且几乎所有细菌的染色体上都存在TA系统。大肠杆菌携带至少29套毒素-抗毒素(TA)系统，但其中只有一小部分的作用机制被破译。已知的毒素抑制广泛的细胞功能，包括DNA、mRNA、30S和50S核糖体亚基和细胞分裂(17、30、37、38)。这些毒素对细菌生理的确切作用尚不清楚。一个可能的作用是在某些严酷的生长条件下调节细胞的生长以求生存(抑菌)。另一种理论认为，毒素具有自杀性，会杀死不需要的细胞，以维持所需的种群(杀菌)，就像在隔离后的杀戮中看到的那样。来自大肠杆菌染色体的ymgG-ymgD和dicB-ydfD操纵子已经被鉴定为可能的TA系统，使用计算机程序预测细菌基因组中的TA系统(RASTA)(27)。YmgD和ydfD都编码一个短肽(分别由109个和65个氨基酸残基组成)，在表达时对细胞有毒性。然而，他们的细胞目标还没有确定。根据我的研究的初步结果，这些毒素的目标似乎是细胞壁，在诱导后导致细胞活力迅速下降。这是TA毒素靶向细胞壁导致细胞死亡的第一个例子。有趣的是，YmgD是由一种信号肽产生的，是第一个被发现分泌到周质空间的毒素。在本申请中，我将尝试破译这些毒素所针对的肽聚糖(PG)生物发生中的确切分子反应。我将提出一个研究项目，研究Ydfd如何降低PG的交联度。当YdfD被诱导表达时，4‘-3’交联键(一个PG茎的4‘-D-丙氨酸残基和另一个的3’Meso-DAP之间)被特异性地减少。我预测YDFDD可能会抑制4‘-3’交联物的形成，从而削弱PG聚合物的交联性，导致细胞溶解。为了揭示YdfD如何降低交联度的机制，我将首先在体外检测系统中检测YdfD的表达对使用纯化蛋白形成聚合物所需的特定酶反应的影响。还将对前体进行层析分析，以检查生物合成中间体的积累/缺乏。YmgD诱导后纯化的肽聚糖缺少大部分的肽聚糖交联脂蛋白。结合这一结果和我们的初步结果，YmgD的毒性在脂蛋白缺陷株中被降低，我们假设YmgD的毒性是脂蛋白依赖的。我们发现YmgD与外膜脂蛋白转运体的两个亚基Lol复合体相互作用。LOL复合体负责将包括LPP在内的外膜脂蛋白从膜内转运到外膜。有90多种外膜脂蛋白是已知的，并具有基本功能。LOL系统的抑制是已知的导致细胞溶解的原因，这是由于脂蛋白在内膜中的积累。YMGD可能通过抑制Lol系统，导致脂蛋白定位不当而发挥毒性作用。我将通过差速离心法从细胞膜中提纯脂蛋白来检验这一假设，并寻找任何错误定位的脂蛋白的异常积累。YmgD对Lol复合体活性的影响也将在体外系统中进行评估。本文提出的研究将促进对不同TA系统控制的复杂细菌生理的理解。由于许多致病细菌还携带许多TA系统，并可能参与致病，因此对TA系统的进一步表征将为开发治疗病原体的新策略提供线索。此外，由于毒素针对细胞壁并引起快速的细胞裂解，我们的研究将直接为我们提供一种新的手段来研究细胞壁生物合成的基础生物学，并将揭示药物设计的新靶点，也将导致开发新的抗生素。