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: 8432004
• 负责人: NILGUN E TUMER
• 金额: $ 19.38万
• 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 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432004
• 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; public health relevance; 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.

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）。他们被归类为 B 类生物恐怖主义特选人员。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，也是有记录以来最致命的一次。 HUS 是美国婴幼儿肾衰竭的最常见原因。没有有效对抗产生这些毒素的细菌感染的具体保护措施或治疗方法。志贺毒素是 AB5 毒素或 II 型核糖体失活蛋白 (RIP) 的一个家族，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，可特异性去除 28S rRNA 的 ¿-sarcin/ricin 环 (SRL) 中的腺嘌呤，从而抑制蛋白质合成。与产生 Stx1 的菌株相比，产生 Stx2 的大肠杆菌菌株更有可能与 HUS 的进展相关。 Stx1 和 Stx2 细胞毒性差异的机制尚不清楚。我们开发了酿酒酵母作为研究 RIP 细胞毒性的强大模型。使用该模型，我们表明核糖体柄对于 Stx1A 和 Stx2A 对 SRL 进行脱嘌呤至关重要。我们的初步数据表明 Stx1A 和 Stx2A 对核糖体柄突变的反应不同。我们首次证明 Stx2 对茎的亲和力比 Stx1 更高。我们建议检查 Stx1 和 Stx2 与来自酵母和哺乳动物细胞的核糖体的相互作用，以检验它们对核糖体柄的需求不同的假设，并且最终它们与核糖体柄的相互作用对于核糖体脱嘌呤和细胞毒性至关重要。我们将确定志贺毒素的核糖体特异性是否是由于它们与茎的相互作用，以及与 Stx2A1 识别序列相对应的肽是否可以阻止毒素活性。识别志贺毒素与核糖体结合的机制差异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。由于导致德国爆发的 Stx2 的 A 和 B 亚基的氨基酸序列与我们实验室正在研究的 Stx2 相同，因此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。