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率和死亡率的重要原因， 尤其是老年人。肺炎链球菌(肺炎球菌)是引起CAP的主要原因。在CAP期间， 肺炎球菌释放溶气素(PLY)和细胞壁成分，破坏肺泡-毛细血管屏障。 肺炎球菌通过侵蚀的血管内皮细胞侵入血流，导致菌血症和败血症。 在25%-30%的患者中，有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。令人遗憾的是，关于肺炎球菌如何 调节破坏肺泡-毛细血管屏障的层和细胞壁成分的释放。我们的长期目标 就是设计有效的治疗方法来对抗肺炎球菌感染。这项建议的直接目标是 阐明能力调节的别溶蛋白在突破空气-血液屏障中的作用并设计出有效的 防止菌血症和败血症的措施。初步来说，我们已经证明了大师ComX的诱导 能力刺激性信息素多肽(CSP)调节能力的调节 别溶酶LytA、CbpD和CibAB的表达。分解作用大大增强了PLE的释放，并且 最终，促进肺炎球菌从感染的肺中逃逸，导致全身性菌血症。更糟糕的是， 被广泛用于治疗肺炎球菌感染的氟喹诺酮类和克拉维酸类抗生素能够 激活COMX依赖的解体作用。值得注意的是，我们综合了显性-负性能力 刺激性多肽(DnCSP)，并显示它们竞争性地抑制COMX依赖的别解反应的诱导， 在细菌性肺炎期间，PLE释放和溶血，并降低小鼠死亡率。我们提出3个具体的建议 目的是验证假设，即dnCSPs将有效地抑制COMX调节的别溶 细菌性肺炎。在目标1中，将使用化学合成和结构分析来推导出下一个 产生高效的dnCSP，将抑制ComX调节的别溶作用。然后，野生型和dnCSP都将 用来确定CSP中涉及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ply和细胞壁的别解释放 细菌性肺炎球菌肺炎期间的成分-在有或没有接受治疗的情况下 氟喹诺酮类药物--导致空气-血液屏障破坏、败血症以及小鼠的心肺功能障碍。在AIM 3，我们将确定dnCSPs在减轻空气-血屏障破坏、细菌性肺炎方面的疗效。 肺炎球菌感染小鼠模型中的败血症以及肺和心功能障碍，在或 未接受氟喹诺酮类药物治疗。该提案的完成将为治疗肺炎球菌开辟新的道路 通过靶向能力调节子进行感染。

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.