A Unique Target for Discovery of Novel Anti-infectives

本研究旨在通过分子遗传学、蛋白质生物化学、基因表达谱分析、蛋白质组学和生物信息学等多学科的综合研究，阐明SmpB.SsrA核糖体拯救系统的分子机制及其在鼠疫耶尔森氏菌和土拉弗朗西斯氏菌的生存、毒力和致病机制中的作用。生物恐怖主义的威胁和越来越多的病原细菌中抗生素耐药性的出现突出了开发新型抗感染剂的需要。现有的抗微生物药物靶向已知的与蛋白质合成、DNA复制、细胞壁和膜合成、细胞增殖、细胞凋亡和细胞凋亡相关的细胞机制。 分裂，或营养吸收和运输。新抗生素的开发需要关注新的靶点，并需要对其细胞功能的基本分子理解。有希望的是，细菌具有独特的靶标-SmpB.SsrA介导的核糖体拯救、蛋白质标记和定向降解系统-被认为参与病原菌在其宿主的敌对微生态位中的适应和存活。这个独特的系统，也被称为反式翻译，是由一个显着的RNA（SsrA RNA）协调的，该RNA既作为tRNA又作为mRNA，以促进在C末端添加短降解标签 新生的多肽。SsrA的所有已知活性都需要SmpB，SmpB是一种特异性结合SsrA的小蛋白，具有高亲和力，以促进其与停滞核糖体的结合。 形成SmpB.SsrA复合物的分子基础、其对受损核糖体的识别以及随后添加的降解标签还不清楚。为了帮助我们在这些研究中，我们将开发一种体外反式翻译系统，使我们能够仔细研究的SmpB-SsrA介导的核糖体救援过程的机制细节。该体外系统也将用作筛选测定，用于选择特异性抑制该过程的小分子。此外，我们希望鉴定和表征参与此过程的其他细胞因子，以用作新的靶标。因为反式翻译系统只存在于原核生物中，并且涉及对于大多数（如果不是全部）致病细菌的生存至关重要的新RNA和蛋白质因子，所以对这种独特过程的更好理解应该允许发现和/或设计高度特异性的新型抗菌剂。

The objective of this research program is to use a combination of molecular genetics, protein biochemistry, global gene expression profiling, proteomics and bioinformatics approaches to elucidate the molecular mechanism of the SmpB.SsrA ribosome rescue system and its role in survival, virulence, and pathogenesis of two category A pathogens, Yersinia pestis and Francisella tularensis. The menace of bioterrorism and the emergence of antibiotic resistance in an increasing number of pathogenic bacterial highlight the need for the development of novel classes of anti-infective agents. Existing antimicrobial drugs target known cellular mechanisms associated with protein synthesis, DNA replication, cell wall and membrane synthesis, cell division, or nutrient uptake and transport. New antibiotic development needs to focus on novel targets and will require a basic molecular understanding of their cellular function. Promisingly, bacteria possess a unique target -the SmpB.SsrA mediated ribosome rescue, protein tagging and directed degradation system- that is thought to participate in the adaptation and survival of pathogenic bacteria in the hostile micro-niches of their hosts. This unique system, also known as trans-translation, is orchestrated by a remarkable RNA (SsrA RNA) that functions both as a tRNA and an mRNA to facilitate the addition of a short degradation tag to the C-terminus of nascent polypeptides. All known activities of SsrA require SmpB, a small protein that binds SsrA specifically and with high affinity to promote its association with stalled ribosomes. The molecular basis for the formation of the SmpB.SsrA complex, its recognition of impaired ribosomes, and the subsequent addition of the degradation tag are not well understood. To assist us in these studies, we will develop an in vitro trans-translation system that should enable us to scrutinize the mechanistic details of the SmpB-SsrA mediated ribosome rescue process. This in vitro system will also be utilized as a screening assay for selection of small molecules that specifically inhibit this process. Furthermore, we wish to identify and characterize additional cellular factors that participate in this process for use as novel targets. Because the trans-translation system exists exclusively in prokaryotes and involves novel RNA and protein factors that are essential for the survival of most (if not all) pathogenic bacteria, a better understanding of this unique process should permit the discovery and/or design of highly specific novel anti-bacterial agents.