Probing the Role of Meso-DAP and Lysine for Growth and Pathogenicity of Pseudomonas aeruginosa.

Antibiotic-resistant bacteria represent a major global challenge as increasing infections become recalcitrant to standard treatments. A lack of novel therapeutics entering the market in the past 30 years further exacerbates this issue and highlights the importance of identifying and validating novel antibiotic targets. In this study, we explored prospective therapeutic targets by examining two metabolites in the lysine biosynthesis pathway, meso-diaminopimelate (DAP) and lysine, within the critically listed pathogen Pseudomonas aeruginosa. These metabolites are involved in bacterial cell wall and protein synthesis; therefore, enzymes present in this pathway represent potential targets for novel therapeutics. To elucidate the validity of these targets, we generated for the first time, gene deletion mutants of the P. aeruginosa DHDPR- and DAPDC-encoding genes using a two-step allelic exchange method. Both the mutants resulted in a lethal phenotype that could be rescued by supplementation with meso-DAP and/or lysine. We subsequently characterized the mutants' pathogenicity in a Galleria mellonella infection model. The DHDPR mutant was unable to provide a lethal infection in this model. Given the importance of these metabolites to membrane and cell wall synthesis, we investigated membrane permeability utilizing a fluorescent probe assay and transmission electron microscopy. Due to their increased membrane permeability, these mutants exhibited greater sensitivity to antibiotics commonly used against Pseudomonas infections. Overall, this study highlights that targeting the lysine biosynthesis pathway could enhance bacterial susceptibility to existing antibiotics, supporting its development as an adjuvant strategy to potentiate current treatments and extend their clinical utility.