莱姆病病原 B. garinii 和 B. afzelii 持留菌的形成机制和药物敏感性研究

Lyme disease, caused by Borrelia spirochete, is the leading tick-borne disease in the world. Currently late Lyme disease with chronic infection and post-treatment Lyme disease syndrome (PTLDS) is difficult to cure with the current Lyme antibiotics. Although the cause of chronic Lyme and PTLDS is unclear, many studies suggest that the current antibiotics are not able to eradicate Borrelia persisters, which may pose a significant problem for effective treatment of persistent Lyme disease. In our previous study, we developed a new viability assay for B. burgdorferi that can be used for high throughput drug screens and identified a number of drug candidates with higher activity against B. burgdorferi persisters than the current Lyme antibiotics. However, we know little about the persisters and drug susceptibility of B. garinii and B. afzelii which are the main Lyme pathogens in China and European instead of B. burgdorferi. Based on our promising data, we would like to develop new viability assay for B. garinii and B. afzelii stains. Based the new assay, this proposal will address the critical issue of whether current clinically used antibiotics are active against B. garinii and B. afzelii and their persisters. Using the new viability assay, we can also build the B. garinii and B. afzelii persister model and identify the drug activity against their persisters by screenning the drug library. According to the known function of identified active drugs, we may explore the mechanism of the formation of B. garinii and B. afzelii persisters. Identified drug candidates also can be used for developing new regimen for the chronic Lyme disease.

莱姆病是一种广泛流行的虫媒传染病。目前慢性莱姆病很难治愈，病原体持留感染是其主要原因之一。伽氏(B. garinii)和埃氏(B. afzeli)疏螺旋体是我国和欧洲莱姆病的主要病原，它们和研究较多的美国莱姆病病原伯氏疏螺旋体有明显差异。目前对伽氏和埃氏疏螺旋体的持留机制和药物敏感性还缺乏系统研究；治疗其持留菌的药物也不明确。因此了解这两种菌的持留机制和发现抗其持留菌的药物是解决我国慢性莱姆病治疗难题的关键。申请人此前对伯氏疏螺旋体的持留和药物敏感性进行过系统研究，发现了与持留菌形成相关的机制，找到了抗其持留菌的有效药物，相关研究得到广泛的关注和认可。本项研究将在此基础上针对伽氏和埃氏疏螺旋体建立有效的活性检测法和持留菌模型，研究其持留菌的特征和药物敏感性，进而寻找对其持留菌有效的治疗药物和药物组合；并将通过有效药物的已知作用机理，研究持留菌形成及耐药的机制，为研究慢性莱姆病提供新的线索。

莱姆病是由广义伯氏疏螺旋体引起的一种蜱传传染病。大多数早期莱姆病患者可以通过一到两周的抗生素治疗治愈，但仍有一些患者患有慢性莱姆病或称治疗后莱姆病综合征(PTLDS)。伯氏疏螺旋体的持留导致对药物不敏感被认为是一个致病因素。寻找靶向伯氏疏螺旋体持留菌的新药对于研究更有效的治疗方法至关重要。目前莱姆病病原主要包括三种，分别是在北美流行的狭义伯氏疏螺旋体、在欧亚流行的伽氏（B. garinii）和埃氏（B. afzelii）螺旋体。此前的研究主要集中于狭义伯氏疏螺旋体，而对在我国流行的伽氏和埃氏螺旋体研究较少。本项目对在我国流行的伽氏和埃氏疏螺旋体优化建立了培养体系和高通灵活性检测方法。针对这两种螺旋体的临床常用药的药敏进行检测和比较研究，发现不同的菌种之间的药物敏感性还是有一定差异，为我国莱姆病的临床治疗提供了科学依据。进一步，我们研究证实了伽氏和埃氏疏螺旋体也可以形成高度广谱耐药的持留菌，为这两种菌导致的慢性感染的机制研究提供了一些科学基础。在以上研究基础上，我们对抗伽氏、埃氏疏螺旋体持留菌的药物开展了高通量筛选。在筛选的2427个化合物中，竹红菌甲素、蒽醌类药物以及其他一些天然化合物和外用药物都展现出超过现有临床用药的杀伽氏和埃氏疏螺旋体持留菌的活力。我们首次发现来源于中国传统中药竹黄的竹红菌甲素是最有效的抗广义伯氏疏螺旋体药物，该药物能够根除稳定期的三种致病广义伯氏疏螺旋体；且对金黄色葡萄球菌的活性较弱，对大肠埃希菌没有活性，是一种特异性抗伯氏疏螺旋体的药物。本项目进一步对竹红菌甲素抗伯氏疏螺旋体的机制进行了研究，通过抗氧化剂拮抗实验，我们明确了竹红菌甲素抗伯氏疏螺旋体的作用机制是结合于伯氏疏螺旋体细胞膜的竹红菌甲素利用光能产生活性氧损伤细胞膜。本项目筛选发现的新药物特别是天然药物有助于研发更有效的莱姆病特别是慢性莱姆病治疗方案，今后我们将进一步进行动物实验等更深入的验证研究。