Role of the ribosomal stalk in the activity of Shiga toxins

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，可导致严重的发病率和死亡率，包括出血性结肠炎（HC）和溶血性尿毒症综合征（HUS）。他们被列为B类生物恐怖主义特效药。最近在德国爆发的产志贺毒素2 （Stx2）大肠杆菌疫情是全球最大的溶血性尿毒综合征疫情之一，也是有记录以来最致命的一次。溶血性尿毒综合征是美国婴幼儿肾衰竭的最常见原因。目前还没有针对产生这些毒素的细菌感染的有效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（RIPs）的一个家族，由酶活性a亚基与相同B亚基的五聚体结合组成。A亚基是一种n -糖苷酶，它特异性地从28S rRNA的-sarcin/ricin环（SRL）中去除腺嘌呤，从而抑制蛋白质合成。产生Stx2的大肠杆菌菌株比产生Stx1的菌株更有可能发展为溶血性尿毒综合征。导致Stx1和Stx2细胞毒性差异的机制尚不清楚。我们已经开发了酵母，酿酒酵母，作为一个强大的模型来研究rip的细胞毒性。通过该模型，我们发现核糖体柄对于Stx1A和Stx2A去纯化SRL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。我们首次发现Stx2比Stx1对茎有更高的亲和力。我们建议研究Stx1和Stx2与酵母和哺乳动物细胞中的核糖体的相互作用，以验证它们对核糖体柄的需求不同，最终它们与柄的相互作用对核糖体去嘌呤和细胞毒性至关重要的假设。我们将确定志贺毒素的核糖体特异性是否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断毒素活性。确定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起作用以及如何阻断其活性提供重要的一步。由于导致德国爆发的Stx2的A和B亚基在氨基酸序列上与我们在实验室研究的Stx2相同，因此拟议的研究与导致世界上最大的溶血性尿毒综合征流行的德国菌株有关。

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.