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

当细菌与先天免疫系统的吞噬细胞相互作用时，它们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，人们知之甚少。然而，最近的工作在威廉姆斯实验室已经确定了一些重要的生化系统的保护机会，医院病原体铜绿假单胞菌对这些巯基反应oxidized.The本项目的目的是：（i）测试的实用性环境质谱技术，快速蒸发电离质谱（REIMS），在了解细菌的应激反应。(i)利用REIMS与其他MS，蛋白质组学，遗传学和生物化学方法相结合，进一步探索铜绿假单胞菌采用的氧化保护系统。快速蒸发电离质谱（REIMS）是一种新型的MS电离方法，由Takats实验室开发，被证明特别适合于细胞膜化学的表征。由于假设氧化剂如HOCl和HOSCN会在细胞外与细菌反应并影响细胞包膜功能，因此建议将REIMS作为研究这种相互作用的工具。该项目的初步目标是利用REIMS分析HOCl/HOSCN存在下的一些铜绿假单胞菌突变体。这些突变体缺乏编码先前鉴定的关键生化保护系统的基因，并且与野生型细菌相比，表现出对HOCl/HOSCN暴露的易感性增加。预计将有重大的方法学发展，因为以这种方式使用REIMS的研究将是全新的。实际考虑因素，如HOCl暴露的程序和细菌引入系统进行分析的形式，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后野生型和突变体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的反应机制。氧化应激的生物标志物，具体到HOCl也可以确定。特别关注将给予外排抗生素泵系统所使用的铜绿假单胞菌。外排泵位于细菌细胞包膜中，将有毒物质排出细胞。缺乏编码外排泵系统的基因的突变体表现出对HOCl和HOSCN暴露的敏感性。REIMS特别适合于铜绿假单胞菌细胞膜的询问，因此被提议作为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。进一步地，有可能引入其他MS技术来验证和扩展REIMS分析的结果。例如，这些可以包括多反应监测（MRM）LC-MS 13 MRM使得质谱法能够用于量化样品中的已知物质。如果REIMS可以识别似乎在氧化应激保护中发挥作用的生物分子，那么了解这些物质在不同氧化剂暴露水平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.