Multifaceted effects of galU deletion on phenotype and virulence of Pseudomonas aeruginosa in vitro and in vivo

Pseudomonas aeruginosa is a widespread Gram-negative opportunistic pathogen in environmental and hospital settings, frequently causing respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disorder (COPD) and ventilator-associated pneumonia. In our previous study, a galU-deleted clinical P. aeruginosa was found to exhibit increased susceptibility to polymyxins. The galU gene plays an important role in the biosynthesis of lipopolysaccharide (LPS) O-antigen. Here, we systematically evaluated the effects of galU deletion on the phenotype and virulence of P. aeruginosa PAO1. A galU deletion mutant was successfully constructed in P. aeruginosa PAO1 by CRISPR/Cas9, and the complementation was accomplished by pUCP18 plasmid carrying wildtype galU. The changes in phenotype, virulence and pathogenicity were systemically studied. The results revealed that knockout of galU led to the loss of O-antigen, which affected growth, virulence and pathogenicity through various ways in P. aeruginosa, and significantly affected the susceptibility of P. aeruginosa to polymyxins. Mechanism study suggested the involvements of quorum sensing, Entner-Doudoroff pathway and tyrosine metabolism, etc on bacterial virulence, antibiotic susceptibility changes after galU deletion. galU and the related pathways may serve as effective targets for treatment of P. aeruginosa infection, providing a theoretical basis for the development of novel antibacterial drugs.