Mechanism of Enzymes Mediating Resistance in Pseudomonas

描述(由申请人提供)：囊性纤维化(CF)患者发生慢性呼吸道感染，感染机会性革兰氏阴性细菌铜绿假单胞菌。无细菌破坏的呼吸道炎症和中性粒细胞渗入是这些感染的特征。最近有研究表明，从囊性纤维化患者中分离的假单胞菌在其脂质A结构上有特定的、毒力相关的修饰。这些修饰包括棕榈酸酯和4-氨基阿拉伯糖的取代，导致对阳离子抗菌肽(CAMP)和多粘菌素(CAMP)的抗性，阳离子抗菌肽是先天性免疫的重要组成部分。负责4-氨基阿拉伯糖脂A生物合成的酶聚集在PmrE和PmrHFIJKLm两个基因座上。除pmrM外，这些基因中的任何一个的突变都会取消4-氨基阿拉伯糖加成类脂A和对cAMP的抗药性。Arna(PmrI)催化UDP-葡萄糖醛酸氧化为UDP-4-酮-阿拉伯糖，这是4-氨基阿拉伯糖脂A生物合成的早期步骤。 抑制4-氨基阿拉伯糖-脂蛋白A的生物合成途径将消除铜绿假单胞菌对抗菌肽的耐药性，从而大大增强宿主对假单胞菌慢性感染的免疫应答。这项建议的具体目标是： 具体目标1：确定Arna的三维结构。 特定目的2：对Arna催化的氧化脱羧基反应进行生化表征。 具体目标3：确定在[Ara4NH4+]-Lipid-A生物合成中两种Arna活性的必要性。 具体目标4：[Ara4NH4]-Lipid-A途径中其他酶的结构测定。 为了提高CF患者的生活质量和生存年龄，开发新的策略来管理他们的肺部感染是至关重要的。鉴于这些感染的慢性性质，预防或消除耐药性是一个根本问题。这项建议的重点是表征介导对环磷酰胺耐药的细菌靶标。

DESCRIPTION (provided by applicant): Patients with Cystic Fibrosis (CF) develop chronic airway infections with the opportunistic gram-negative bacteria Pseudomonas aeruginosa. Airway inflammation and neutrophilic infiltration without bacterial destruction characterize these infections. It has been recently shown that Pseudomonas isolated from Cystic Fibrosis patients have specific, virulence-associated modifications in their lipid A structure. These modifications, which include substitutions with palmitate and 4-aminoarabinose, are responsible for resistance to cationic antimicrobial peptides (CAMPs), an important component of innate immunity and Polymyxin, a CAMP antibiotic. The enzymes responsible for the biosynthesis of 4-aminoarabinose-lipid A are clustered in two loci termed PmrE and PmrHFIJKLM. Mutation of any of these genes except pmrM abolishes 4-aminoarabinose addition to lipid A and resistance to CAMPs. ArnA (PmrI) catalyzes the oxidation of UDP-Glucuronic acid to UDP-4-keto-arabinose, an early step in the proposed biosynthesis of 4- aminoarabinose-lipid A. Inhibition of the pathway for 4-aminoarabinose-lipid A biosynthesis would abolish Pseudomonas aeruginosa resistance to antimicrobial peptides, therefore greatly enhancing the host immune response against chronic infections with Pseudomonas. The specific aims of this proposal are: Specific Aim 1: Determine the three dimensional structure of ArnA. Specific Aim 2: Biochemically characterize the oxidative decarboxylation reaction catalyzed by ArnA. Specific Aim 3: Establish the need for both ArnA activities in [Ara4NH4+]-Lipid-A biosynthesis. Specific Aim 4: Structure determinations of other enzymes in the [Ara4NH4¿]-Lipid-A pathway. To improve both the quality of life and the survival age of CF patients it is crucial that new strategies are developed to manage their pulmonary infections. Given the chronic nature of these infections preventing or abolishing resistance is a fundamental problem. This proposal focuses on the characterization of bacterial targets that mediate resistance to CAMPs.