Role of the ribosomal stalk in the activity of Shiga toxins

描述(申请人提供)：志贺毒素(STX)产生大肠杆菌(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)。他们被归类为B类生物恐怖主义精选特工。最近在德国爆发的志贺毒素2(Stx2)产生的大肠杆菌是世界上最大的HUS疫情之一，也是有记录以来最致命的疫情。HUS是美国婴幼儿肾功能衰竭最常见的原因。没有特别的保护措施或治疗方法有效地防止产生这些毒素的细菌感染。志贺毒素是一类AB5毒素或II型核糖体失活蛋白(RIPs)，由一个具有酶活性的A亚基组成，它与一个由相同B亚基组成的五聚体相关联。A亚基是一种N-糖苷酶，它特异性地从28S rRNA的sarcin/ricin环(SRL)中移除腺嘌呤，导致蛋白质合成受到抑制。产生STX2的大肠杆菌菌株比产生STX1的菌株更有可能与HUS的进展有关。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RIPS的细胞毒性。利用这个模型，我们证明了核糖体的茎对于Stx1A和Stx2A提纯SRL是至关重要的。我们的初步数据表明，Stx1A和Stx2A对核糖体茎中的突变反应不同。我们首次证明了Stx2对茎的亲和力高于Stx1。我们建议研究STX1和Stx2与来自酵母和哺乳动物细胞的核糖体的相互作用，以检验这两个基因对核糖体茎的需求不同的假设，最终它们与茎的相互作用对于核糖体提纯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由于它们与茎的相互作用，以及对应于Stx2A1识别序列的多肽是否可以阻断毒素活性。识别志贺毒素与核糖体结合的机制差异将为理解这些毒素的工作原理以及如何阻止它们的活性提供重要的一步。由于导致德国疫情的Stx2的A和B亚基在氨基酸序列上与我们实验室正在研究的Stx2相同，因此拟议的研究与导致世界上最大规模HUS疫情的德国毒株有关。

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.