Effects of deletion mutations in aguA on fitness and survival of Pseudomonas aeruginosa cystic fibrosis isolates

Abstract

Pseudomonas aeruginosa is a pathogen commonly associated with the disease cystic fibrosis (CF) due to the bacterium’s ability to adapt to numerous environments. These adaptations include mutations that benefit the pathogen such as antibiotic resistance and host immune evasion. Five P. aeruginosa isolates from CF sputum were found to have deletion mutations in aguA that encodes the agmatine deiminase for this species. These mutations cause the cationic molecule agmatine to accumulate both within and outside the cell. The purpose of this thesis was to determine the effects of agmatine accumulation on fitness and survival of P. aeruginosa in the context of CF. Compared to the isogenic strains with a native aguA, the mutations led to decreased susceptibility to cationic antibiotics (aminoglycosides and polymyxins). At sub-minimal inhibitory concentrations (MIC) of these antibiotics, growth of the aguA+ strains was delayed and stunted more than the aguA mutants. This growth phenotype at these sub-MICs was also seen when Mg2+ and Ca2+ concentrations in the growth medium were reduced. In an acute pneumonia the aguA mutant recruited fewer neutrophils to the murine lungs relative to the aguA+ strain. When testing the ex vivo bronchial epithelial cell response to LPS, agmatine caused reduced IL-8 production in response to LPS in a dose-dependent manner, with the higher concentration of agmatine (100 μM) eliciting a response similar to the negative control without LPS. Together, these data show that the mutations in aguA that prevent the bacteria from breaking down agmatine may be beneficial to the P. aeruginosa isolates while colonizing the CF airways.