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: 8201620
• 负责人: Hisako Masuda
• 金额: $ 5.13万
• 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 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8201620
• 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. PUBLIC HEALTH RELEVANCE: Despite the increasing threat of emerging pathogens and multiple drug resistant pathogenic strains, the discovery of new classes of therapeutic methods has been struggling to keep pace. There is an urgent need for novel antibiotics with new targets. We have identified two novel toxins from E. coli that cause 99 percent of cells to die within 30 min. These toxins appear to compromise cell wall integrity, thus causing cells to undergo cell death, and in one case, leads to lysis. In this application, I will propose to investigate the mechanisms of how toxins interfere with cell wall integrity and their potential as a therapeutic means to cure pathogenic bacteria. The outcome of this study will provide important insights into new approaches for development of novel antibiotics.

描述（由申请人提供）：细菌通常被认为是一种单细胞、自由的生物体，但它们的基因组上携带毒素或自杀基因。这些毒素通常与其同源抗毒素共表达。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“分离后杀伤”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，而且存在于几乎所有细菌的染色体中。E.大肠杆菌携带至少29套毒素-抗毒素（TA）系统，但只有其中一小部分的作用机制被破译。已知毒素抑制广泛的细胞功能，包括DNA、mRNA、30 S和50 S核糖体亚基和细胞分裂（17，30，37，38）。这些毒素对细菌生理学的确切作用尚未清楚了解。一个可能的作用是在某些苛刻的生长条件下调节细胞生长以存活（抑菌）。另一种理论是毒素是自杀性的，杀死不需要的细胞以维持所需的群体（杀菌），如在分离后杀死中所见（6）。ymgG-ymgD和dicB-MifD操纵子来自E.已经使用计算机程序预测细菌基因组中的TA系统（RASTA）将大肠杆菌染色体鉴定为可能的TA系统（27）。ymgD和MyfD都编码短肽（分别由109和65个氨基酸残基组成），其在表达时对细胞有毒。然而，它们的细胞靶点尚未确定。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是与信号肽一起产生的，并且是发现分泌到周质空间中的第一种毒素。在这个应用程序中，我将试图破译肽聚糖（PG）的生物合成，这些毒素的目标确切的分子反应。我将提出一个研究项目，研究YdfD如何降低PG交联的程度。当诱导YdfD表达时，特异性地减少4 '-3'交联（一个PG茎的4'D-丙氨酸残基与另一个的3'内消旋-DAP之间）。我预测YdfD可以抑制4 '-3'交联的形成，从而削弱PG聚合物的交联，导致细胞裂解。为了揭示YdfD如何减少交联的机制，我将首先使用纯化的蛋白质在体外测定系统中研究YdfD表达对聚合物形成所需的特异性酶促反应的影响。还将对前体进行色谱分析，以检查生物合成中间体的积累/缺乏。在YmgD诱导后纯化的肽聚糖缺乏大部分肽聚糖交联脂蛋白。结合这一结果与我们的初步结果，YmgD毒性降低的脂蛋白缺乏的菌株，我们假设，YmgD毒性是脂蛋白依赖性的。我们发现YmgD与外膜脂蛋白转运蛋白Lol复合物的两个亚基相互作用。Lol复合物负责将外膜脂蛋白（包括Lpp）从膜内转运到外膜。已知有90多种外膜脂蛋白具有重要功能。已知Lol系统的抑制由于脂蛋白在内膜中的积累而引起细胞溶解。YmgD可能通过抑制Lol系统，引起脂蛋白的不适当定位而发挥其毒性。我将通过差速离心法从细胞膜中纯化脂蛋白来检验这一假设，并寻找任何不寻常的错误定位的脂蛋白积累。还将使用体外系统评估YmgD对Lol复合物活性的影响。这里提出的研究将推进对由各种TA系统控制的复杂细菌生理学的理解。由于许多病原菌也携带许多TA系统，并可能参与致病性，TA系统的进一步表征将提供线索，以开发一种新的策略来治疗病原体。此外，由于毒素靶向细胞壁并导致快速细胞裂解，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，还将导致开发新的抗生素。 公共卫生关系：尽管新兴病原体和多重耐药致病菌株的威胁越来越大，但新类别的治疗方法的发现一直在努力跟上步伐。迫切需要具有新靶点的新型抗生素。从大肠杆菌中鉴定出两种新毒素。这些毒素似乎损害细胞壁的完整性，从而导致细胞经历细胞死亡，在一种情况下，导致细胞溶解。在本申请中，我将提议研究毒素如何干扰细胞壁完整性的机制及其作为治疗病原菌的治疗手段的潜力。这项研究的结果将为开发新型抗生素的新方法提供重要的见解。