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）和下氨酸酸（HOSCN）。关于细菌如何保护自己免受HOCL和HOSCN的影响知之甚少。然而，威廉姆斯实验室的最新工作已经确定了许多在保护机会性的铜绿假单胞菌针对这些硫醇反应性氧化剂的生物化学系统。 (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.由于假设HOCL和HOSCN等氧化剂将在细菌外反应和影响细胞包膜功能，因此已提出Reims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在HOCL/HOSCN的存在下分析A eruginosa突变体。与野生型细菌相比，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，并证明对HOCL/HOSCN暴露的敏感性增加。预计有重大的方法论发展是通过这种方式采用这种方式的研究将完全新颖。实用的考虑因素，例如HOCL暴露过程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。然后，下一步将是分析产生的光谱数据，并在暴露于HOCL时确定野生型和突变体质谱之间的任何差异。假设这种差异可能会阐明细菌反应对氧化剂的机制。还可以鉴定出特定于HOCL的氧化应激的生物标记物。外排泵位于细菌细胞包膜中，并从细胞中输出有毒物质。缺乏编码外排泵系统基因的突变体对HOCL和HOSCN暴露表现出敏感性。 Reims特别适合对铜绿假单胞菌细胞膜的询问，因此被提出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清洁的缺失突变体。从线下，有可能引入其他MS技术来验证和扩展Reims分析的结果。例如，这些可能包括多个反应监测（MRM）LC-MS。 MRM使质谱法可以用于量化样品中已知物种。如果Reims可以识别似乎在氧化应激保护中起作用的生物分子，那么了解这些物种在不同水平的氧化剂暴露水平的浓度就可以进一步了解它们在系统中的确切作用。