Controlling bactermic pneumococcal pneumonia with synthetic dominant-negative competence peptides

项目摘要 社区获得性肺炎（CAP）是所有年龄段患者发病和死亡的重要原因， 尤其是老年人。肺炎链球菌（肺炎球菌）是CAP的主要病因。在CAP期间， 肺炎球菌释放肺炎球菌溶血素（pneumolysin，pneumolysin）和细胞壁成分，破坏肺泡-毛细血管屏障。 肺炎球菌通过被侵蚀的血管内皮侵入血流，引起菌血症和败血症 25 - 30%的患者，5 - 7%的死亡率。其他并发症包括多器官功能障碍，脓胸， 呼吸衰竭、心包炎和不良心脏事件。遗憾的是，人们对肺炎球菌 调节破坏肺泡-毛细血管屏障的细胞壁成分的释放。我们的长期目标 是设计有效治疗肺炎球菌感染的方法。这项建议的近期目标是 阐明能力调节的同种异体在突破气-血屏障中的作用，并设计有效的 防止菌血症和败血症的措施。初步，我们已经证明了主程序ComX的归纳 感受态刺激信息素肽（CSP）的感受态调节子的调节剂，上调 同种异型分解酶LytA、CbpD和CibAB的表达。同种异体溶解显著增强了β-淀粉样蛋白的释放， 最终促使肺炎球菌从感染的肺部逃逸，引起全身性菌血症。更糟糕的是， 广泛用于治疗肺炎球菌感染的氟喹诺酮类和克拉维霉素类抗生素能够 激活ComX依赖的同种异体溶解值得注意的是，我们已经合成了显性-阴性能力 刺激肽（dnCSPs），并显示它们竞争性抑制ComX依赖性同种异体溶解的诱导， 降低细菌性肺炎时小鼠的死亡率。我们提出3个具体的 目的是检验dnCSPs将有效抑制ComX调节的同种异体溶解的假设， 菌血症性肺炎在目标1中，化学合成和结构分析将用于推导下一个 产生将抑制ComX调节的同种异体溶解的高效力dnCSP。然后，野生型和dnCSP都将 可用于鉴定CSP中参与结合和激活的关键结构特征， 组氨酸激酶受体ComD。在目标2中，我们将检查ComX是否诱导了细胞壁和细胞壁的同种异体释放， 在菌血症性肺炎球菌肺炎期间的成分-在存在或不存在以下治疗的情况下 氟喹诺酮类药物在小鼠中导致空气-血液屏障破坏、败血症以及肺和心脏功能障碍。在aim中 3，我们将确定dnCSP在减弱空气-血液屏障破坏、菌血症性肺炎 和脓毒症，以及肺和心脏功能障碍的小鼠模型中的肺炎球菌感染，在存在或 没有氟喹诺酮治疗。该提案的完成将为治疗肺炎球菌开辟新途径 通过靶向感受态调节子进行感染。

Project Summary Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality in patients of all ages, particularly the elderly. Streptococcus pneumoniae (pneumococcus) is a major cause of CAP. During CAP, pneumococcus releases pneumolysin (PLY) and cell wall components, which disrupt alveolar-capillary barrier. Pneumococcus invades bloodstream through the eroded vascular endothelium, causing bacteremia and sepsis in 25-30% of patients, with 5-7% fatality. Other complications include multi-organ dysfunction, empyema, respiratory failure, pericarditis, and adverse cardiac events. Regretfully, little is known about how pneumococcus regulates the release of PLY and cell wall components that disrupt alveolar-capillary barrier. Our long-term goal is to device effective therapy against pneumococcal infection. The immediate objectives of this proposal are to elucidate the role of competence-regulated allolyis in breaching the air-blood barrier and devise effective measures to thwart bacteremia and sepsis. Preliminarily, we have shown that induction of ComX, the master regulator of competence regulon by the competence stimulating pheromone peptide (CSP), upregulates the expression of allolytic enzymes LytA, CbpD and CibAB. Allolysis substantially enhances the release of PLY, and ultimately, facilitates pneumococcal escape from infected lung to cause systemic bacteremia. Worse yet, fluoroquinolone and clavulanate antibiotics widely prescribed to treat pneumococcal infection are capable of activating ComX-dependent allolysis. Significantly, we have synthesized dominant-negative competence stimulating peptides (dnCSPs) and shown they competitively inhibit the induction of ComX-dependent allolysis, PLY release and hemolysis, and reduce mouse mortality during bacteremic pneumonia. We propose 3 Specific Aims to test the hypothesis that dnCSPs will be effective in inhibiting the ComX-regulated allolysis during bacteremic pneumonia. In Aim 1, chemical synthesis and structural analysis will be used to derive the next generation high potency dnCSPs that will inhibit ComX-regulated allolysis. Then, both wild-type and dnCSPs will be utilized to identify the key structural features in CSP that are involved in both binding and activating of the histidine kinase receptor ComD. In Aim 2, we will examine if ComX-induced allolytic release of PLY and cell wall components during bacteremic pneumococcal pneumonia—in the presence or absence of treatment with fluoroquinolones—lead to air-blood barrier disruption, sepsis, and lung and cardiac dysfunction in mice. In Aim 3, we will determine the efficacy of dnCSPs in attenuating air-blood barrier disruption, bacteremic pneumonia and sepsis, and lung and cardiac dysfunction in mouse model of pneumococcal infection, in the presence or absence of fluoroquinolone treatment. Completion of the proposal will chart new path to treat pneumococcal infection by targeting the competence regulon.