Understanding the response of Pseudomonas aeruginosa to hypochlorous and hypothiocyanous acid using Rapid Evaporative Ionisation Mass Spectrometry

Bacteria encounter the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) when they interact with phagocytic cells of the innate immune system. Little is known about how bacteria protect themselves against HOCl and HOSCN. However, recent work in the Williams Lab has identified a number of biochemical systems important in the protection of the opportunistic, nosocomial pathogen Pseudomonas aeruginosa against these thiol-reactive oxidants.

The aims of this project are:(i) test the utility of ambient mass spectrometry technique, Rapid evaporative ionisation mass spectrometry (REIMS), in understanding bacterial stress responses. (i) to exploit REIMS in conjunction with other MS, proteomic, genetic and biochemical approaches to further explore the oxidative protection systems employed by P. aeruginosa.

Rapid evaporative ionisation mass spectrometry (REIMS) is a novel ionisation method for MS, developed in the Takats lab, proven to be particularly suited for the characterisation of cell membrane chemistry. As it is hypothesised that oxidants like HOCl and HOSCN will react extracellularly with bacteria and impact cell envelope function, REIMS has been proposed as a tool for investigation into this interaction.

The initial aim of the project is to utilise REIMS to analyse a number P. aeruginosa mutants in the presence of HOCl/HOSCN. These mutants lack genes coding for previously identified key biochemical protection systems and demonstrate increased susceptibility to HOCl/HOSCN exposure, compared to wildtype bacteria. Significant methodological development is anticipated as such a study employing REIMS in this fashion would be entirely novel. Practical considerations such as the procedure for HOCl exposure and the format in which the bacteria are introduced to the system for analysis will greatly influence the data produced.

The next step will then be to analyse the spectral data produced and identify any differences between wildtype and mutant mass spectra upon exposure to HOCl. It is hypothesised that such disparities may shed light on the mechanisms involved in the bacterial response to the oxidant. Biomarkers of oxidative stress specific to HOCl may also be identified.

Particular focus will be given to the efflux antibiotic pump systems utilised by P. aeruginosa. Efflux pumps sit in the bacterial cell envelope and export toxic substances out of the cell. Mutants lacking the genes coding for efflux pump systems demonstrate sensitivity to HOCl and HOSCN exposure. REIMS is particularly suited for interrogation of the P. aeruginosa cell membrane and therefore is proposed as a potentially useful tool for further investigation into the role of these pumps in oxidative stress protection. To achieve this, the first stage will be to generate clean deletion mutants in these genes of interest.

Further down the line, there is potential to introduce other MS techniques to verify and expand upon the results of REIMS analysis. For example, these could include multiple reaction monitoring (MRM) LC-MS. MRM enables mass spectrometry to be used to quantify known species within a sample. If REIMS can identify biomolecules that appear to play a role in oxidative stress protection, knowing the concentration of these species at different levels of oxidant exposure provide further insight in to their exact role in the system.