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

使用快速蒸发电离质谱了解铜绿假单胞菌对次氯酸和次硫氰酸的反应

• 批准号: 2131276
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2131276
• 关键词: Understanding; response; Pseudomonas; aeruginosa; hypochlorous

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.

当细菌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，人们知之甚少。然而，威廉姆斯实验室最近的工作已经确定了许多生化系统，这些系统对于保护机会性院内病原体铜绿假单胞菌免受这些硫醇反应性氧化剂的侵害非常重要。该项目的目的是：（i）测试环境质量的效用光谱技术，快速蒸发电离质谱（REIMS），用于了解细菌应激反应。 (i) 将 REIMS 与其他 MS、蛋白质组学、遗传和生化方法结合使用，进一步探索铜绿假单胞菌使用的氧化保护系统。快速蒸发电离质谱 (REIMS) 是一种新型的 MS 电离方法，在Takats 实验室被证明特别适合细胞膜化学表征。由于假设 HOCl 和 HOSCN 等氧化剂会与细菌发生细胞外反应并影响细胞包膜功能，因此 REIMS 被提议作为研究这种相互作用的工具。该项目的最初目标是利用 REIMS 分析一些 P。 HOCl/HOSCN 存在下的铜绿假单胞菌突变体。这些突变体缺乏编码先前确定的关键生化保护系统的基因，并且与野生型细菌相比，对 HOCl/HOSCN 暴露的敏感性增加。预计方法学将取得重大进展，因为以这种方式采用 REIMS 的研究将是全新的。实际考虑因素（例如次氯酸暴露的程序以及将细菌引入系统进行分析的格式）将极大地影响生成的数据。下一步将是分析产生的光谱数据，并识别暴露于次氯酸后野生型和突变体质谱之间的任何差异。据推测，这种差异可能有助于揭示细菌对氧化剂反应的机制。还可以鉴定 HOCl 特有的氧化应激生物标志物。将特别关注铜绿假单胞菌利用的外排抗生素泵系统。外排泵位于细菌细胞包膜中，将有毒物质排出细胞外。缺乏外排泵系统编码基因的突变体表现出对 HOCl 和 HOSCN 暴露的敏感性。 REIMS 特别适合检测铜绿假单胞菌细胞膜，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，有可能引入其他 MS 技术来验证和扩展 REIMS 分析的结果。例如，这些可能包括多反应监测 (MRM) LC-MS。 MRM 使质谱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。