Administrative Supplement: Borrelia gene products critical for natural infection cycle

项目摘要/摘要 莱姆病是世界上许多地区最流行的扁虱传播感染，包括美国 每年新增病例超过45万例。这种疾病是由一组细菌病原体疏螺旋体引起的 伯格多费氏菌在自然界中通过涉及硬蜱的复杂的地方性感染循环而茁壮成长 肩部扁虱和各种脊椎动物宿主。尽管付出了很多努力，但感染仍然很难控制， 很大程度上是由于缺乏包括疫苗在内的预防战略，早期诊断困难 感染，以及未能使用目前的抗生素实现彻底治愈。具体地说，几个月后 标准护理抗生素治疗的一部分患者可能会经历一系列持续或复发 症状，称为慢性莱姆病或治疗后莱姆病综合征(PTLDS)。 而细菌感染的持续性可能有助于或可能不有助于 对于PTLDS，其治疗方案仍不清楚。因此，疫苗和新药的开发是 完全有根据。我们已经确定了一种名为BbHtrA的关键毒力决定因素，它在 螺旋体在宿主感染性和持久性中的作用。蛋白质的缺失使病原体不再是 在哺乳动物宿主中具有传染性。基于这一信息，我们提出了一种治疗策略，依赖于 小分子药物对蛋白质功能的干扰。为此，我们将识别小分子， 采用高通量筛选(HTS)的方法阻断BbHtrA蛋白水解酶活性 基于纳米DSF和荧光酶抑制技术的化合物文库。此屏幕的点击量 将通过二次细胞测试进行验证，揭示出可以渗透到B细胞的有效化合物。 伯格多费尔膜。将与国家研究中心合作进行化合物筛选 高级翻译科学(NCATS)调查人员，他们提供专业知识、基础设施和现代 化合物文库，并在HTS和药物发现方面拥有丰富的经验。我们还将尝试 开展BbHtrA的结构研究，这对开发新的治疗药物将是重要的 以蛋白质为目标。在莱姆病的整个动物模型中对选定的HIT分子的验证将是 在未来更大规模的赠款申请中进行审查。该项目采用了尖端技术，目标是 相互作用的毒力决定因素来对抗莱姆病，这是一种可能避免PTLDS的新策略。 此外，同样的方法也可以作为防治其他硬虱传播感染的范例，包括 那些是由最近发现的更具毒性的莱姆病病原体菌株引起的。

PROJECT SUMMARY/ABSTRACT Lyme disease is the most prevalent tick-borne infection in many parts of the world, including the U.S., where over 450,000 new cases occur annually. The disease is inflicted by a group of bacterial pathogens, Borrelia burgdorferi sensu lato, that thrive in nature through a complex enzootic infection cycle involving Ixodes scapularis ticks and a variety of vertebrate hosts. Despite much effort, the infection remains difficult to control, largely due to the absence of preventive strategies including vaccines, difficulties in the diagnosis of early infection, and failure to achieve complete cures using current antibiotics. Specifically, several months after standard-care antibiotic therapy, a subset of patients can experience a series of persistent or relapsing symptoms, which are termed as chronic Lyme disease or post-treatment Lyme disease syndrome (PTLDS). While the persistence of the bacterial infection may or may not contribute to the etiology or pathogenesis of PTLDS, its treatment options remain unknown. Therefore, the development of vaccines and new drugs is highly warranted. We have identified a critical virulence determinant called BbHtrA that plays an indispensable role in host infectivity and persistence of spirochetes. Deletion of the protein renders the pathogen non- infectious in mammalian hosts. Based on this information, we propose a therapeutics strategy that relies on disruption of the protein function by small molecule drugs. To this end, we will identify small molecules that disrupt BbHtrA protease activity by employing an assay designed for high-throughput screening (HTS) of large compound libraries based on nanoDSF and fluorescence protease inhibition technology. Hits of this screen will be validated by a secondary cellular assay, revealing potent compounds that are permeable across the B. burgdorferi membrane. Compound screening will be performed in collaboration with National Center for Advancing Translational Sciences (NCATS) investigators, who provide expertise, infrastructure, and modern compound libraries, and have extensive experience with HTS and drug discovery. We will also attempt to develop structural studies on BbHtrA, which will be important for the development of new therapeutic agents targeting the protein. The validation of selected hit molecules in a whole animal model of Lyme disease will be examined in future larger grant applications. The project employs cutting-edge technology that targets interacting virulence determinants to combat Lyme disease, a novel strategy that may avoid PTLDS. Moreover, the same approach may serve as a paradigm for combating other tick-borne infections, including those caused by the recently-discovered, more virulent strains of Lyme disease pathogens.