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: 8713792
• 负责人: Hisako Masuda
• 金额: $ 0.3万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 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/8713792
• 关键词: Characterization; new; toxins; YmgD; YdfD

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（由申请人提供）：细菌通常被认为是单细胞的、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是由单个操纵子编码的，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，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，而且还存在于几乎所有细菌的染色体中。大肠杆菌携带至少 29 套毒素-抗毒素 (TA) 系统，但其作用机制仅在少数情况下被破译。 他们。已知的毒素会抑制多种细胞功能，包括 DNA、mRNA、30S 和 50S 核糖体亚基以及细胞分裂 (17、30、37、38)。这些毒素对细菌生理学的确切作用尚不清楚。一种可能的作用是在某些严格的生长条件下调节细胞生长以维持生存（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 维持所需的种群数量（杀菌），如隔离后杀戮中所见 (6)。使用计算机程序预测细菌基因组中的 TA 系统 (RASTA)，已将来自大肠杆菌染色体的 ymgG-ymgD 和 dicB-ydfD 操纵子确定为可能的 TA 系统 (27)。 ymgD 和 ydfD 均编码短肽（由 109 个和 65 个氨基酸组成） 酸残基），它们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，导致细胞活力在诱导后迅速下降。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 是由信号肽产生的，是第一个发现的毒素 分泌到周质空间。在此应用中，我将尝试破译这些毒素针对的肽聚糖 (PG) 生物合成中的确切分子反应。我将提出一个研究项目，研究 YdfD 如何降低 PG 交联度。当 YdfD 表达被诱导时，4'-3' 交联（在一个 PG 茎的 4' D-丙氨酸残基和 3' 另一种的内消旋DAP）被专门还原。我预测YdfD可能会抑制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，我将首先检查 YdfD 表达对使用纯化的聚合物形成所需的特定酶反应的影响 体外测定系统中的蛋白质。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。 YmgD诱导后纯化的肽聚糖缺乏大部分肽聚糖交联脂蛋白。将此结果与我们的初步结果（脂蛋白缺陷菌株中 YmgD 毒性降低）相结合，我们 假设 YmgD 毒性是脂蛋白依赖性的。我们发现 YmgD 与外膜脂蛋白易位子 Lol 复合物的两个亚基相互作用。 Lol 复合体负责将外膜脂蛋白（包括 Lpp）从内膜转运至外膜。已知有 90 多种外膜脂蛋白具有重要功能。 Lol系统的抑制 已知由于脂蛋白在内膜中的积累而导致细胞裂解。 YmgD可能通过抑制Lol系统发挥毒性，导致脂蛋白定位不当。我将通过差速离心从膜中纯化脂蛋白来检验这一假设，并寻找错误定位脂蛋白的任何异常积累。 YmgD对活性的影响 Lol 复合物的含量也将使用体外系统进行评估。这里提出的研究将促进对各种 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大量的TA系统，并且可能参与致病性，因此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，由于毒素靶向细胞壁并导致细胞快速裂解，我们的研究将直接为我们提供一种新的方法 研究细胞壁生物合成的基础生物学，将揭示药物设计的新目标，并导致新型抗生素的开发。