Anti-Pseudomonas Biofilm Therapy for Cystic Fibrosis

描述（由申请人提供）：CF是一种常染色体隐性遗传疾病，在美国约有30，000例患者，在全球约有60，000例患者，平均预期寿命约为30岁。铜绿假单胞菌肺部感染是囊性纤维化（CF）患者死亡的主要原因。这种细菌通过产生一种叫做藻酸盐的胞外多糖来建立生物膜。生物膜的生长保护生物体免受抗生素和宿主防御机制的侵害。一般来说，这些慢性感染并没有根除，尽管积极的抗生素治疗，因此需要新的治疗方法。这项提议将开发一种新的疗法，可以破坏生物膜并杀死细菌。藻酸裂解酶（AL）将与杀菌肽CAP 18化学连接。这种缀合物对藻酸盐的消化将向细菌递送细胞毒性CAP 18肽，并增加其对抗生素的敏感性和宿主防御机制。此外，由于CAP 18肽可以结合并中和脂多糖（LPS），因此另外的益处将是抑制导致CF患者肺功能丧失的炎症。该提案将克隆、表达和纯化重组AL，然后将其化学连接到来自CAP 18的有效杀菌合成肽。将在几项体外试验中检测偶联物AL-CAP 18，以验证融合物仍保留各组分的功能活性。这将包括LPS的中和、杀菌活性、AL酶活性、通过藻酸盐促进抗生素扩散以及促进嗜中性粒细胞吞噬和杀死铜绿假单胞菌。将评价AL-CAP 18在CF患者痰液存在下发挥作用的能力，以降低粘度和杀菌作用。将研究AL-CAP 18与常规抗生素和重组DNA酶增强或协同的潜力。这种新的疗法将提供一种杀菌剂，可以杀死耐药细菌，并结合一种酶，破坏生物膜的结构。这种治疗应该加强常规抗生素和天然宿主防御机制渗透到生物膜中，从而获得细菌。目前，还没有批准的药物可以溶解细菌藻酸盐，因此AL-CAP 18代表了一种具有独特作用机制的新疗法，使CF患者受益。

DESCRIPTION (provided by applicant): CF is an autosomal recessive genetic disorder affecting approximately 30,000 patients in the United States and 60,000 patients worldwide, with an average life expectancy of about 30 years. Lung infection with Pseudomonas aeruginosa is a leading cause of mortality in cystic fibrosis (CF) patients. The bacteria establish biofilms through the production of an exopolysaccharide called alginate. Growth in biofilms protects the organism from antibiotics and host defense mechanisms. Generally these chronic infections are not eradicated despite aggressive antibiotic therapy, thus new treatments are needed. This proposal will develop a novel therapy that will disrupt the biofilm and kill the bacteria. Alginate lyase (AL) will be chemically linked to the bactericidal peptide, CAP18. Digestion of the alginate by this conjugate will deliver the cytotoxic CAP18 peptide to the bacteria, as well as increase its susceptibility to antibiotics and host defense mechanisms. Furthermore, since CAP18 peptides can bind and neutralize lipopolysaccharide (LPS), an additional benefit will be inhibition of inflammation which contributes to loss of pulmonary function in CF patients. This proposal will clone, express, and purify recombinant AL which will then be chemically linked to a potently bactericidal synthetic peptide derived from CAP18. The conjugate, AL-CAP18 will be tested in several in vitro assays to verify the fusion still retains the functional activity of each component. This will include neutralization of LPS, bactericidal activity, AL enzyme activity, promotion of antibiotic diffusion through alginate, and promotion of neutrophil phagocytosis and killing of P. aeruginosa. The ability of AL-CAP18 to function in the presence of sputum from CF patients will be evaluated with respect to reduction of viscosity and bactericidal effects. The potential of AL-CAP18 to augment or synergize with conventional antibiotics and recombinant DNase will be investigated. This novel therapy will provide a bactericidal agent that can kill antibiotic-resistant bacteria combined with an enzyme that will disrupt the architecture of the biofilm. This treatment should potentiate the penetration of conventional antibiotics and natural host defense mechanisms into the biofilm to thus gain access to the bacteria. Currently, there is no approved drug that can dissolve bacterial alginate, thus AL-CAP18 represents a new therapy with a unique mechanism of action to benefit CF patients.