Pseudomonas aeruginosa is an opportunistic pathogen, responsible for life-threatening infections in immunocompromised people. To establish chronic infections, P. aeruginosa must survive exposure to antimicrobial oxidants generated by the innate immune system, however, the mechanisms by which this is achieved are not well understood. The aim of this thesis project was to complement previous genetic and transcriptomic studies by employing mass spectrometry (MS) to address this knowledge gap.
Laser assisted rapid evaporative ionisation mass spectrometry (LA-REIMS) was proposed as a tool for metabolic profiling of P. aeruginosa exposed to oxidative stress. A novel protocol for untargeted, high-throughput and automated LA-REIMS analysis of bacterial samples was developed. The protocol proved able to reproducibly detect differences between untreated P. aeruginosa samples and samples exposed to either hypochlorous acid (HOCl), hypothiocyanous acid (HOSCN), hydrogen peroxide (H2O2), methylglyoxal (MGO) or the superoxide generator methyl viologen (MV). Samples were classified according to their oxidative stress exposure with 90 - 100 \% accuracy and a panel of 54 biomarkers of oxidative stress was identified, including alkylquinolones (AQs), rhamnolipids and phospholipids. Biomarkers of HOCl and HOSCN stress were found to be conserved in P. aeruginosa clinical isolates. HOCl and H2O2 were found to cause a significant decrease in the concentration of AQs and rhamnolipids respectively, both important classes of molecule for P. aeruginosa virulence and therefore indicating that the immune system may employ HOCl and H2O2 to target these molecules and reduce P. aeruginosa pathogenicity. Indeed, the concentrations of pyocyanin and elastase, two virulence factors regulated by the AQ-mediated Pseudomonas Quinolone Signal (PQS) system, were found to be markedly depleted in HOCl-treated samples. Previous studies showed that the multidrug efflux system MexEF-OprN is upregulated in response to HOCl and HOSCN stress. An LC-MS approach was developed to identify potential substrates exported by the efflux system in response to HOCl stress and a panel of candidate features was generated for future characterisation.