Role of the ribosomal stalk in the activity of Shiga toxins

性状（由申请方提供）：产滋贺毒素（Stx）的E.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）和溶血性尿毒综合征（HUS）。它们被列为B类生物恐怖主义选择剂。最近爆发了产滋贺毒素2（Stx 2）的大肠杆菌。德国爆发的HUS是世界上最大的HUS爆发之一，也是有记录以来最致命的。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有效的治疗方法来对抗产生这些毒素的细菌的感染。滋贺毒素是AB 5毒素或II型核糖体失活蛋白（RIP）的一个家族，由与相同B亚基的五聚体缔合的酶活性A亚基组成。A亚基是一种N-糖苷酶，可特异性地从28 S rRNA的<$-八叠球菌素/蓖麻毒素环（SRL）中去除腺嘌呤，从而抑制蛋白质合成。E.与Stx 1菌株相比，Stx 2大肠杆菌菌株更可能与HUS进展相关。解释Stx 1和Stx 2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。利用这个模型，我们发现核糖体柄是关键的Stx 1A和Stx 2A去嘌呤的SRL。我们的初步数据表明，Stx 1A和Stx 2A对核糖体柄突变的反应不同。我们首次表明，Stx 2具有更高的亲和力比Stx 1的柄。我们建议检查Stx 1和Stx 2与酵母和哺乳动物细胞的核糖体的相互作用，以测试假设，它们在核糖体柄的要求不同，最终它们与柄的相互作用是核糖体脱嘌呤和细胞毒性的关键。我们将确定滋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Stx 2A 1的识别序列对应的肽是否可以阻断毒素活性。确定滋贺毒素与核糖体结合的机制差异将为理解这些毒素如何工作以及如何阻止其活性迈出重要一步。由于导致德国疫情的Stx 2的A和B亚基与我们实验室正在研究的Stx 2的氨基酸序列相同，因此拟议的研究与导致世界上最大的HUS流行的德国菌株有关。 公共卫生相关性：产滋贺毒素E.大肠杆菌菌株是食源性病原体，可引起严重甚至致命的影响。没有有效的解毒剂或治疗剂对抗滋贺毒素介导的溶血性尿毒综合征（HUS）。我们建议了解滋贺毒素如何与核糖体相互作用，以开发针对E.大肠杆菌中毒

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.