Developing a Novel Plague Antibiotic by Targeting Protein Secretion

描述（由申请人提供）：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，鼠疫发病率、鼠疫流行地区的范围和数量稳步增加，最令人不安的是，首次出现了对用于治疗鼠疫的大多数抗生素具有广泛耐药性的菌株。再加上需要立即有效的治疗，耐药菌株的出现尤其令人不安，并强调需要更好地了解这种细菌可能具有的任何独特易感性，以及利用这种易感性的分子。我们最近观察到Y.鼠疫菌对芳基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，这些抗生素通过一种新的机制起作用，即抑制细菌酶信号肽酶I（SPase），从而抑制蛋白质分泌，这需要SPase。事实上，专性寄生虫Y.鼠疫菌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。这种明显的超敏反应支持了Y染色体对蛋白质分泌的依赖性。鼠疫，这表明靶向分泌的抗生素可能对治疗鼠疫特别有用。我们将试图确定Y超敏反应的起源。通过四个目标将鼠疫菌转化为芳香霉素。首先，将使用生物化学和遗传学方法来确定Y。鼠疫菌SPase以异常低的水平产生，或者芳基霉素是否以异常高的亲和力结合它（Aim 1）。接下来，转录谱将用于确定Y。鼠疫菌缺乏启动应激反应，而其他细菌利用应激反应更好地耐受由芳基霉素介导的蛋白质分泌应激（Aim 2）。然后我们将使用2D-LC/MS/MS来确定Y.鼠疫菌的超敏反应是由于抑制了特定蛋白质的分泌而引起的，这些蛋白质要么形成有毒中间体，要么是获取营养所必需的（目标3）。最后，对Y.将通过使用全基因组重测序来确定鼠疫菌对芳霉素的抗性，以表征体外进化为抗性的克隆（目的4）。这种方法将首次提供芳基霉素作为抗鼠疫抗生素的详细分析，并应揭示其独特的抗Y的生物化学起源。鼠疫活动。此外，结果应确定是否Y。鼠疫对蛋白质分泌的抑制剂特别敏感，这对我们理解鼠疫的生理学和如何开发治疗鼠疫的抗生素具有重要意义。 公共卫生相关性：鼠疫耶尔森氏菌（鼠疫的致病菌）抗药性的出现，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于蛋白质分泌来提供毒素和营养素，我们发现它对一类新型天然产物过敏，已知这些天然产物会抑制其他细菌的蛋白质分泌。我们建议描述超敏反应的起源，以验证抑制蛋白质分泌作为一种新的，特别有前途的方法来开发药物来对抗这种古老但重新出现的疾病。

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.