Role of the ribosomal stalk in the activity of Shiga toxins

核糖体柄在志贺毒素活性中的作用

基本信息

批准号：
8303644
负责人：
NILGUN E TUMER
金额：
$ 22.49万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8303644
关键词：
Accounting Adenine Affect Affinity Amino Acid Sequence Antidotes Archaea Bacteria Binding Bioterrorism Categories Cells Characteristics Child Cleaved cell Colitis Complex Cytosol Data Defect Depurination Disease Outbreaks Elements Endoplasmic Reticulum Epidemic Epidemiology Escherichia coli Escherichia coli EHEC Escherichia coli O157 Exotoxins Family Family member German population Germany Glycolipids Hemolytic-Uremic Syndrome Human Infant Infection Kidney Failure Kinetics Mammalian Cell Measures Mediating Modeling Morbidity - disease rate Mutation N glycosidase Organism Pathogenesis Pathogenicity Penetration Peptides Poisoning Protein Synthesis Inhibition Proteins Public Health Relative (related person)Ribosomal RNA Ribosomes Ricin Ricin A Chain Role Saccharomyces Safety Shiga Toxin Shiga-Like Toxin I Shiga-Like Toxin II Specificity Structure Testing Therapeutic Time Toxin Virulence Factors Work Yeasts cytotoxicity dimer enteroaggregative Escherichia coli foodborne pathogen globotriaosylceramide holotoxins in vivo mortality mutant shiga toxin 2 B subunit

项目摘要

DESCRIPTION (provided by applicant): Shiga toxin (Stx) producing E. coli (STEC) are foodborne pathogens that can cause severe morbidity and mortality, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). They are classified as category B bioterrorism select agents. A recent outbreak of Shiga toxin 2 (Stx2) producing E. coli in Germany represented one of the largest outbreaks of HUS worldwide and was the deadliest on record. HUS is the most common cause of renal failure in infants and young children in the US. There are no specific protective measures or therapeutics effective against infection by bacteria producing these toxins. Shiga toxins are a family of AB5 toxins or type II ribosome-inactivating proteins (RIPs), consisting of an enzymatically active A subunit that associates with a pentamer of identical B subunits. The A subunit is an N-glycosidase that specifically removes an adenine from the ¿-sarcin/ricin loop (SRL) of 28S rRNA, resulting in inhibition of protein synthesis. E. coli strains producing Stx2 are more likely to be associated with progression to HUS than strains producing Stx1. The mechanism that accounts for the differences in cytotoxicity of Stx1 and Stx2 is not known. We have developed the yeast, Saccharomyces cerevisae, as a powerful model to study the cytotoxicity of RIPs. Using this model, we showed that ribosomal stalk is critical for Stx1A and Stx2A to depurinate the SRL. Our preliminary data indicates that Stx1A and Stx2A respond differently to mutations in the ribosomal stalk. We show for the first time that Stx2 has higher affinity for the stalk than Stx1. We propose to examine the interaction of Stx1 and Stx2 with ribosomes from yeast and mammalian cells to test the hypothesis that they differ in their requirements for the ribosomal stalk and ultimately their interaction with the stalk is critical for ribosome depurination and cytotoxicity. We will determine if ribosome specificity of Shiga toxins is due to their interactions with the stalk and if peptides corresponding to the recognition sequences of Stx2A1 can block toxin activity. Identifying the mechanistic differences in binding of Shiga toxins to ribosomes would provide a major step towards understanding how these toxins work and how to block their activity. Since the A and the B subunits of Stx2 responsible for the German outbreak are identical in amino acid sequence to the Stx2 we are studying in our lab, the proposed studies are relevant to the German strain that caused the largest HUS epidemic in the world. PUBLIC HEALTH RELEVANCE: Shiga toxin producing E. coli strains are foodborne pathogens that can cause serious and sometimes fatal effects. There are no antidotes or therapeutics effective against Shiga toxin-mediated hemolytic uremic syndrome (HUS). We propose to understand how Shiga toxins interact with ribosomes to develop remedies against E. coli poisoning.

描述（由申请人提供）：产生志贺毒素（Stx）的大肠杆菌（STEC）是食源性病原体，可导致严重的发病和死亡，包括出血性结肠炎（HC）和溶血性尿毒症综合征（HUS），它们被归类为 B 类。德国最近爆发的产志贺毒素 2 (Stx2) 大肠杆菌是全球最大规模的 HUS 爆发之一。 HUS 是美国婴儿和幼儿肾衰竭的最常见原因，目前尚无针对产生这些毒素（AB5 毒素或 II 型）的细菌感染有效的具体保护措施或治疗方法。核糖体失活蛋白 (RIP)，由与相同 B 亚基的五聚体结合的酶活性 A 亚基组成。A 亚基是一种 N-糖苷酶，特异性地作用。从 ¿ 中去除腺嘌呤28S rRNA 的 sarcin/ricin 环 (SRL)，导致蛋白质合成受到抑制，与产生 Stx1 的菌株相比，产生 Stx2 的大肠杆菌菌株更可能与 HUS 的进展相关。 Stx1 和 Stx2 未知。我们开发了酿酒酵母作为研究 RIP 细胞毒性的强大模型。核糖体柄对于 Stx1A 和 Stx2A 脱嘌呤至关重要。我们的初步数据表明，Stx1A 和 Stx2A 对核糖体柄突变的反应不同，我们首次表明 Stx2 对核糖体柄的亲和力高于 Stx1。检查 Stx1 和 Stx2 与酵母和哺乳动物细胞核糖体的相互作用，以检验它们对核糖体柄的需求不同以及最终相互作用的假设我们将确定志贺毒素的核糖体特异性是否是由于其与茎的相互作用，以及与 Stx2A1 识别序列相对应的肽是否可以阻断毒素活性。由于 Stx2 的 A 和 B 亚基负责产生志贺毒素，因此核糖体的志贺毒素将为了解这些毒素如何发挥作用以及如何阻止其活性迈出重要一步。德国爆发的病毒与我们实验室正在研究的 Stx2 的氨基酸序列相同，拟议的研究与引起世界上最大规模 HUS 流行的德国菌株相关。公共健康相关性：产生志贺毒素的大肠杆菌菌株是食源性病原体，可造成严重甚至致命的影响，目前尚无有效对抗志贺毒素介导的溶血性尿毒症综合征 (HUS) 的解毒剂或治疗方法。与核糖体一起开发针对大肠杆菌中毒的治疗方法。