A Unique Target for Discovery of Novel Anti-infectives

本研究的目的是结合分子遗传学、蛋白质生物化学、全球基因表达谱、蛋白质组学和生物信息学的方法，阐明SmpB.SsrA核糖体拯救系统的分子机制及其在两种A类病原体--鼠疫耶尔森氏菌和图拉氏方济氏菌的生存、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菌对抗生素产生耐药性，凸显了开发新型抗感染药物的必要性。现有的抗菌药物针对已知的与蛋白质合成、DNA复制、细胞壁和膜合成、细胞 分裂，或养分的吸收和运输。新抗生素的开发需要专注于新的靶点，并需要对其细胞功能有基本的分子理解。有希望的是，细菌拥有一个独特的靶标-SmpB.SsrA介导的核糖体拯救、蛋白质标记和定向降解系统-被认为参与了病原菌在宿主敌意微生态位中的适应和生存。这个独特的系统，也被称为反式翻译，是由一个非凡的RNA(SsrA RNA)协调的，它既具有tRNA的功能，又具有mRNA的功能，以促进在C末端增加一个短的降解标签 新生的多肽。所有已知的SsrA活性都需要SmpB，SmpB是一种小蛋白，可以与SsrA特异结合，并具有高亲和力，以促进其与停滞的核糖体的结合。 SmpB.SsrA复合体的形成、其对受损核糖体的识别以及随后添加的降解标签的分子基础尚不清楚。为了帮助我们进行这些研究，我们将开发一个体外反式翻译系统，使我们能够仔细检查SmpB-SsrA介导的核糖体拯救过程的机制细节。这个体外系统也将被用作选择特定抑制这一过程的小分子的筛选试验。此外，我们希望确定和表征参与这一过程的其他细胞因子，以用作新的靶点。由于反式翻译系统仅存在于原核生物中，并涉及对大多数(如果不是全部)致病菌的生存至关重要的新的RNA和蛋白质因子，因此对这一独特过程的更好理解应该有助于发现和/或设计高度特异的新型抗菌剂。

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.