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

描述（由申请人提供）：细菌通常被认为是一个单细胞，自由活的有机体，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，并且需要“隔离后杀伤”（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上。大肠杆菌携带至少29组毒素-抗毒素（TA）系统，但其作用机制仅在其中一小部分被破译。已知毒素可抑制多种细胞功能，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生长以维持生存（抑菌）。另一种理论是，毒素是自杀的，杀死不需要的细胞以维持所需的种群（杀菌），如种族隔离后的杀戮(6)。利用计算机程序预测细菌基因组中的TA系统（RASTA），大肠杆菌染色体上的ymgG-ymgD和dicB-ydfD操纵子已被确定为可能的TA系统（27）。ymgD和ydfD编码一个短肽（分别由109和65个氨基酸残基组成），表达后对细胞具有毒性。然而，它们的细胞靶点尚未确定。根据我的初步研究结果，这些毒素的目标似乎是细胞壁，在它们的诱导下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质周间隙的毒素。在这个应用中，我将试图破译肽聚糖（PG）生物发生的确切分子反应，这些毒素的目标。我将提出一个研究项目，研究YdfD如何降低PG交联的程度。当YdfD表达被诱导时，4‘-3’交联（一个PG茎的4' d -丙氨酸残基与另一个PG茎的3' meso-DAP之间的交联）特异性减少。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，我将首先在体外分析系统中使用纯化蛋白质检查YdfD表达对聚合物形成所需的特定酶反应的影响。还将进行前体的色谱分析，以检查生物合成中间体的积累/缺乏。诱导YmgD后纯化的肽聚糖缺乏大部分肽聚糖交联脂蛋白。将这一结果与我们在脂蛋白缺乏菌株中YmgD毒性降低的初步结果相结合，我们假设YmgD毒性是脂蛋白依赖的。我们发现YmgD与外膜脂蛋白转运体Lol复合物的两个亚基相互作用。Lol复合物负责外膜脂蛋白（包括Lpp）从膜内转运到外膜。已知的外膜脂蛋白有90多种，具有重要的功能。已知Lol系统的抑制会引起细胞裂解，这是由于内膜中脂蛋白的积累。YmgD可能通过抑制Lol系统发挥毒性作用，导致脂蛋白定位不正确。我将通过差速离心从膜中纯化脂蛋白来验证这一假设，并寻找任何不寻常的错定位脂蛋白积累。我们还将利用体外系统评估YmgD对Lol复合物活性的影响。本文提出的研究将促进对各种TA系统控制的复杂细菌生理的理解。由于许多致病菌也携带许多TA系统，并可能参与致病性，因此进一步表征TA系统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引起细胞快速裂解，我们的研究将直接为我们研究细胞壁生物合成的基础生物学提供新的手段，并将揭示药物设计的新靶点，也将导致开发新的抗生素。

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.