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

细菌在与先天免疫系统的吞噬细胞相互作用时，会遇到氧化剂次氯酸（HOCl）和次硫氰酸（HOSCN）。关于细菌如何保护自己免受HOCl和HOSCN的侵害，我们知之甚少。然而，威廉姆斯实验室最近的工作已经确定了一些生物化学系统，这些系统在保护机会性的、医院内的病原体铜绿假单胞菌免受这些硫醇反应性氧化剂的伤害方面很重要。该项目的目的是：(i)测试环境质谱技术，快速蒸发电离质谱（REIMS）在理解细菌应激反应方面的效用。(i)利用REIMS与其他质谱、蛋白质组学、遗传学和生化方法的结合，进一步探索铜绿假单胞菌的氧化保护系统。快速蒸发电离质谱（REIMS）是Takats实验室开发的一种新的质谱电离方法，被证明特别适合于细胞膜化学的表征。由于假设HOCl和HOSCN等氧化剂会在细胞外与细菌发生反应并影响细胞包膜功能，因此提出了rems作为研究这种相互作用的工具。该项目的最初目的是利用REIMS分析存在HOCl/HOSCN的铜绿假单胞菌突变体。这些突变体缺乏先前确定的关键生化保护系统的编码基因，并且与野生型细菌相比，对HOCl/HOSCN暴露的易感性增加。预期会有重大的方法发展，因为以这种方式采用REIMS的研究将是完全新颖的。实际考虑因素，如HOCl暴露的程序和将细菌引入系统进行分析的格式，将极大地影响所产生的数据。下一步将是分析产生的光谱数据，并确定暴露于HOCl后野生型和突变型质谱之间的任何差异。据推测，这种差异可能揭示了细菌对氧化剂反应的机制。氧化应激特异性HOCl的生物标志物也可能被确定。将特别关注铜绿假单胞菌使用的外排抗生素泵系统。外排泵位于细菌细胞包膜内，将有毒物质排出细胞。缺乏外排泵系统编码基因的突变体对HOCl和HOSCN暴露敏感。rems特别适合于对铜绿假单胞菌细胞膜的询问，因此被认为是进一步研究这些泵在氧化应激保护中的作用的潜在有用工具。为了实现这一目标，第一阶段将是在这些感兴趣的基因中产生干净的缺失突变体。今后，有可能引入其他质谱技术来验证和扩展REIMS分析的结果。例如，这些可能包括多重反应监测（MRM） LC-MS。MRM使质谱法能够用于定量样品中的已知物种。如果REIMS能够识别出在氧化应激保护中发挥作用的生物分子，那么了解这些物种在不同水平的氧化剂暴露下的浓度可以进一步了解它们在系统中的确切作用。

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.