Developing a Novel Plague Antibiotic by Targeting Protein Secretion

描述(申请人提供)：鼠疫耶尔森氏菌是鼠疫的病原体，是古代和中世纪所有细菌病原体中最致命的。然而，近年来，在鼠疫流行地区的范围和数量上，鼠疫发病率稳步上升，最可怕的是，对大多数用于治疗鼠疫的抗生素具有广泛抗药性的菌株首次具有广泛的抗药性。再加上需要立即进行有效的治疗，耐药菌株的出现尤其令人不安，并突显出需要更好地了解这种细菌可能具有的任何独特的敏感性，以及利用这种敏感性的分子。我们最近观察到鼠疫耶尔森氏菌对芳香霉素类天然产物抗生素过敏。在其他细菌中，我们和其他人已经证明了这些抗生素通过一种新的机制发挥作用，即抑制细菌酶信号肽I(Spase)，从而抑制蛋白质分泌，这是Spase所必需的。事实上，专性寄生虫鼠疫杆菌可能高度依赖蛋白质分泌来组装建立感染和从环境中清除营养所需的系统。这种明显的过敏性支持了鼠疫杆菌对蛋白质分泌的更强依赖，这表明针对分泌的抗生素可能对治疗鼠疫特别有用。我们将通过四个目标来确定鼠疫耶尔森氏菌对芳香菌素类药物过敏的起源。首先，将使用生化和遗传学方法来确定鼠疫杆菌Spase的产生水平是否异常低，或者芳香菌素是否以异常高的亲和力结合它(目标1)。接下来，将使用转录图谱来确定鼠疫杆菌是否在启动应激反应方面存在缺陷，其他细菌使用这些应激反应来更好地耐受芳香菌素介导的蛋白质分泌应激(目标2)。然后，我们将使用2D-LC/MS/MS来确定鼠疫杆菌过敏是否是由于抑制特定蛋白质的分泌所致，这些蛋白质形成有毒中间体或营养获取所需的蛋白质(目标3)。最后，将通过使用全基因组重新测序来确定鼠疫耶尔森氏菌在体外进化为抗药性克隆的机制(目标4)。这种方法将首次详细分析芳香菌素作为抗鼠疫抗生素，并将揭示其独特的抗Y-Y的生化来源。鼠疫活跃性。此外，这些结果应该确定鼠疫杆菌是否特别容易受到蛋白质分泌抑制剂的影响，这对我们理解其生理学以及如何开发抗生素治疗它具有基本的意义。 公共卫生相关性：鼠疫的病原菌--鼠疫耶尔森氏菌出现抗药性，带来了人类历史上最具破坏性的疾病将卷土重来的威胁。作为一种专性寄生虫，鼠疫耶尔森氏菌依赖蛋白质分泌来传递毒素和清除营养物质，我们发现它对一类新的天然产物高度敏感，这些天然产物已知能抑制其他细菌的蛋白质分泌。我们建议对这种超敏反应的起源进行表征，以努力验证抑制蛋白质分泌是一种新的、特别有希望的方法来开发治疗这种古老但复发的疾病的药物。

DESCRIPTION (provided by applicant): Yersinia pestis, the causative agent of plague, was the most deadly of all the bacterial pathogens in ancient and medieval times. However, in recent years there has been a steady increase in plague morbidity, in the extent and number of plague-endemic areas, and most frighteningly, for the first time strains possessing broad resistance to most antibiotics use to treat plague. Combined with the need for immediate effective treatment, the emergence of resistant strains is especially troubling and highlights the need for an improved understanding of any unique susceptibilities that this bacterium might have, and for molecules that take advantage of the susceptibility. We have recently observed that Y. pestis is hypersensitive to the arylomycin class of natural product antibiotics. In other bacteria, we and others have demonstrated that these antibiotics act via a novel mechanism, namely the inhibition of the bacterial enzyme signal peptidase I (SPase), and thus the inhibition of protein secretion, for which SPase is required. Indeed, it is possible that the obligate parasite Y. pestis is hyperdependent on protein secretion to assemble the systems required for the establishing infections and for scavenging nutrients from its environment. The distinct hypersensitivity supports a stronger reliance on protein secretion for viability in Y. pestis, suggesting that antibiotics targeting secretion might be of particular utility for treating plague. We will seek to define the origins of the hypersensitivity of Y. pestis to the arylomycins via four Aims. First, biochemical and genetic methods will be used to determine whether Y. pestis SPase is produced at unusually low levels or whether the arylomycins bind it with an unusually high affinity (Aim 1). Next, transcriptional profiling will be used to determine whether Y. pestis is deficient in initiating stress responses used by other bacteria to better tolerate the protein secretion stress mediated by the arylomycins (Aim 2). We will then use 2D-LC/MS/MS to determine if the Y. pestis hypersensitivity results from inhibiting the secretion of specific proteins, either that form toxic intermediates or that are required for nutrient acquisition (Aim 3). Lastly, the mechanism by which Y. pestis evolves resistance to the arylomycins will be determined by using whole genome re-sequencing to characterize clones evolved to be resistant in vitro (Aim 4). This approach will provide the first detailed analysis of the arylomycins as anti-plague antibiotics and should reveal the biochemical origins of their unique anti-Y. pestis activity. In addition, the results should determine whether Y. pestis is particularly susceptible to inhibitors of protein secretion, with fundamental implications for our understanding of its physiology and for how antibiotics might be developed for its treatment. PUBLIC HEALTH RELEVANCE: The emergence of drug resistance in Yersinia pestis, the causative bacterium of plague, brings with it the threat that the most devastating disease in human history will return. As an obligate parasite, Yersinia pestis relies on protein secretion to deliver toxins and scavenge nutrients, and we have found that it is hypersensitive to a novel class of natural products that are known to inhibit protein secretion in other bacteria. We propose to characterize the origins of the hypersensitivity, in an effort to validate the inhibition of protein secretion as a new and especially promising approach to developing drugs to combat this ancient but re-emergent disease.