C5a in defense against murine Gram-negative pneumonia

描述（由申请人提供）： 该项目正在研究过敏毒素C5 a，一种由免疫系统在第一次与入侵微生物相互作用时产生的蛋白质，如何在急性革兰氏阴性肺炎期间帮助支持肺部的宿主防御。这种疾病对住院、手术后和免疫功能低下的患者构成严重威胁，导致这种疾病的细菌对广谱抗生素的耐药性越来越强。使用肺部感染的小鼠模型，我们的目标是更好地定义C5 a的作用，以便开发出针对革兰氏阴性肺炎的改进疗法。该项目的第一个目的是研究C5 a如何增强肺泡巨噬细胞对临床重要病原体铜绿假单胞菌的体外反应，并将测量C5 a对吞噬作用，呼吸爆发，细菌杀伤和促炎介质释放的影响。平行研究将检查C5 a在全血中对这种病原体的作用。第二个目标将研究革兰氏阴性菌表面的结构如何改变C5 a的产生并最终改变其有效性。这些研究利用了克雷伯氏肺炎菌强毒株的突变体，其中负责启动表面碳水化合物O-抗原合成的基因已被删除。将比较野生型和突变株促进C5 a生成和改变肺泡巨噬细胞反应的能力。将使用E.大肠杆菌，该大肠杆菌已经被转化以合成克雷伯氏菌O-抗原，以确定该补体对抗物的表达是否可以将毒力传递给其他非致病性细菌。该提案的最终目的是研究3种策略，以提高急性感染期间肺部产生的C5 a水平。这些实验将检查用来自眼镜蛇毒液的蛋白质的肠内治疗，所述蛋白质可以在不存在入侵病原体的情况下产生C5 a，羧肽酶-N（C5 a的重要失活剂）的抑制剂，以及最后的重组鼠C5 a。希望这些结果将更好地定义C5 a在革兰氏阴性肺部感染期间宿主防御中的作用，将增加对革兰氏阴性生物如何逃避补体介导的肺部防御的理解，并将确定新型C5 a增强疗法是否可用于在急性细菌性肺炎期间协助宿主防御。

DESCRIPTION (provided by applicant): This project is studying how the anaphylatoxin C5a, a protein generated by the immune system upon first interacting with invading microorganisms, helps support host defenses in the lung during acute Gram negative pneumonia. This disease is a serious threat to hospitalized, post-operative, and immunocompromised patients and the bacteria that cause it are increasingly resistant to broad-spectrum antibiotics. Using a murine model of lung infection, our goal is to better define the role of C5a so that improved therapies against Gram-negative pneumonia might be developed. The first aim of the project examines how C5a enhances the in vitro responses of alveolar macrophages to the clinically important pathogen Pseudomonas aeruginosa and will measure C5a's effect on phagocytosis, respiratory burst, bacterial killing, and release of proinflammatory mediators. Parallel studies will examine C5a's effect against this pathogen in whole blood. The second aim will study how structures on the surface of Gram-negative bacteria may alter the generation and ultimately the effectiveness of C5a. These studies take advantage of a mutant of a virulent strain of Klebsiella pneumoniae in which the gene responsible for initiating synthesis of the surface carbohydrate O-antigen has been deleted. Wild-type and mutant strains will be compared in terms of their ability to promote the generation of C5a and to alter the responses of alveolar macrophages. Parallel studies will be performed using a strain of E. coli which has been transformed to synthesize the Klebsiella O-antigen to determine if expression of this complement countermeasure can convey virulence to an otherwise nonpathogenic bacteria. The final aim of the proposal will examine 3 strategies to boost the level of C5a produced in the lung during acute infection. These experiments will examine intratracheal therapy with a protein derived from cobra venom which can generate C5a in the absence of an invading pathogen, an inhibitor of the enzyme carboxypeptidase-N (an important deactivator of C5a), and lastly recombinant murine C5a. It is hoped that the results will better define the role of C5a in host defense during Gram-negative lung infection, will increase understanding of how Gram-negative organisms evade complement-mediated lung defenses, and will determine whether novel C5a-enhancing therapies might be used to assist host defense during acute bacterial pneumonia.