Developing a Novel Plague Antibiotic by Targeting Protein Secretion

描述（申请人提供）：鼠疫耶尔森氏菌是古代和中世纪最致命的细菌性病原体，是鼠疫的病原体。然而，近年来，鼠疫发病率、鼠疫流行地区的范围和数量稳步上升，最可怕的是，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。再加上需要立即进行有效治疗，耐药菌株的出现尤其令人不安，并突出表明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杆菌对天然产物抗生素中阿霉素类过敏。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，而SPase是必需的。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物质所需的系统。这种明显的超敏反应支持鼠疫耶尔森氏菌更依赖蛋白质分泌来维持生存，这表明针对蛋白质分泌的抗生素可能对治疗鼠疫特别有用。我们将试图通过四个目标来定义鼠疫杆菌对芳霉素过敏的起源。首先，将使用生化和遗传方法来确定鼠疫杆菌SPase是否以异常低的水平产生，还是芳霉素以异常高的亲和力与它结合（目的1）。接下来，转录谱分析将用于确定鼠疫杆菌是否缺乏启动其他细菌用来更好地耐受由芳霉素介导的蛋白质分泌应激的应激反应（目的2）。然后，我们将使用2D-LC/MS/MS来确定鼠疫杆菌的超敏性是否源于抑制特定蛋白质的分泌，无论是形成有毒中间体还是营养获取所需的蛋白质（目标3）。最后，利用全基因组重测序来鉴定在体外进化出耐药性的克隆，将确定鼠疫杆菌进化出对芳霉素耐药的机制（目的4）。该方法将首次对作为抗鼠疫抗生素的芳霉素进行详细的分析，并揭示其独特的抗y抗体的生化起源。耶尔的活动。此外，研究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，这对我们了解其生理学以及如何开发抗生素治疗其具有根本意义。

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.