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. ? ?
