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

• 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
• 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

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（由适用提供）：产生大肠杆菌（STEC）的志贺毒素（STX）是食源性病原体，可能导致严重的发病率和死亡率，包括出血性大肠杆菌（HC）和溶血性尿毒症综合征（HUS）。它们被归类为B类生物恐怖主义选择的代理。最近在德国生产大肠杆菌的Shiga Toxin 2（STX2）爆发是全球HUS爆发之一，是最致命的记录。 HUS是美国婴儿和幼儿肾衰竭的最常见原因。没有特定的保护措施或治疗剂可通过产生这些毒素的细菌有效地感染感染。 Shiga毒素是AB5毒素的家族或II型核糖体灭活蛋白（RIP），由酶活性的A亚基组成，该亚基与相同的B亚基的五聚体相关联。 A亚基是一种N-糖苷酶，它专门从28S rRNA的 - 撒尔生蛋白/ricin环（SRL）中去除腺嘌呤，从而抑制蛋白质合成。与产生STX1的菌株相比，产生STX2的大肠杆菌菌株更可能与进展为HUS。尚不清楚说明STX1和STX2细胞毒性差异的机制。我们已经开发了酵母酿酒酵母，它是研究撕裂细胞毒性的强大模型。使用此模型，我们表明核糖体茎对于STX1A和STX2A部署SRL至关重要。我们的初步数据表明，STX1A和STX2A对核糖体茎突变的反应不同。我们首次表明STX2对茎的亲和力高于STX1。我们建议检查STX1和STX2与酵母和哺乳动物细胞的核糖体的相互作用，以检验其对核糖体茎的要求有所不同，并最终与茎的相互作用对核糖体部署和细胞毒性至关重要。我们将确定Shiga毒素的核糖体特异性是否是由于它们与茎的相互作用引起的，并且Petides是否对应于STX2A1的识别序列可以阻止毒素活性。确定志贺毒素与核糖体的结合机械差异将为了解这些毒素的工作原理以及如何阻止其活性提供一个重要的一步。由于负责德国爆发的STX2的A和B亚基与我们在实验室中研究的STX2相同，因此拟议的研究与导致世界上最大的HUS流行病的德国菌株有关。 公共卫生相关性：产生大肠杆菌菌株的志贺毒素是食源性病原体，可能会引起严重，有时是致命的影响。没有针对志贺毒素介导的溶血性尿毒症综合征（HUS）有效的解毒剂或治疗剂。我们建议了解Shiga毒素如何与核糖体相互作用，以开发针对大肠杆菌中毒的疗法。