How does low-temperature plasma damage the bacterial outer membrane?

全球性挑战包括微生物感染以及表面(例如食物)和环境的细菌污染。随着对抗生素的耐药性和人们日益关注，需要开发替代方法。其中之一是低温等离子体(LTP)。当高压施加到气流中时，会形成生物活性的反应性氧和氮物种的混合物。常压LTP的化学特性为对抗与伤口和皮肤溃疡相关的局部细菌感染提供了巨大的希望，作为一种不含抗生素的疗法，也是我们对抗抗菌素耐药性(AMR)的另一种潜在武器。然而，仍有一些关键的生物学和物理问题需要解决，以促进LTP在社会上的应用。例如，LTP中的活性物质破坏细菌被膜和呈现细胞不活的物理机制是非常不清楚的(例如，膜上是否形成了孔？)为了进一步开发和优化LTP的使用，需要更好地了解其杀菌作用的细胞机制，这将是本项目的重点。我们目前对LTP如何影响微生物的了解主要基于种群水平的细胞活力分析或单细胞成像技术，由于成像前需要广泛的样品准备，因此时间分辨率较低。通过结合我们在实时单细胞荧光成像(CGB)、LTP生成(DO‘C)以及不同时间和长度尺度(MvdW)的细菌表型方面的专业知识，可以在理解等离子体处理如何改变体内和体外的膜双层方面取得重大进展。这些知识将被纳入约克血浆研究所正在进行的工作，该工作的重点是表征和操纵血浆中活性物种的组成，以提高生物活性。这个项目将使用单细胞水平的方法，调查不同的LTP如何影响具有不同细胞表面的革兰氏阴性细菌菌株的细胞膜。学生将利用荧光标记有机磷和脂多糖的方法来监测接触LTP期间和之后细胞包膜的形态变化。单细胞成像将使用先进的多色荧光显微镜方法和开发的新技术，以允许同时暴露于不同类型和剂量的LTP。在暴露于LTP后，将进行延时细胞成像，以确定是否可以观察到任何形态差异，并识别非活细胞和活细胞的潜在表型特征。从这个项目中获得的知识将被用来开发一个LTP诱导的膜损伤的机制模型，并确定可以用来增强血浆对革兰氏阴性菌破坏作用的潜在制剂。这个项目将提供优秀的尖端单细胞分析方法方面的专业培训，并结合对膜生物学和微生物学的良好理解。专业技能培训将由白玫瑰DTP提供。学生还将通过参加导师实验室会议、部门研讨会和研究会议以及参加外展活动来提高专业技能。学生将加入一个充满活力的交叉学科的博士生社区。这个项目适合申请者具有强大的物理和化学背景，并对了解分子水平的生物过程和参与新的抗菌方法的开发有浓厚的兴趣。

Global challenges include microbial infections, and bacterial contamination ofsurfaces (e.g. food) and the environment. With resistance to antibiotics anincreasing concern alternative approaches need to be developed. One of theseis low-temperature plasma (LTP). A mix of biologically active reactive oxygenand nitrogen species are formed when high voltage is applied to a gas flow. Thechemical properties of atmospheric pressure LTP offers great promise as anantibiotic-free therapeutic for combating topical bacterial infection associatedwith wounds and skin ulcers, and are another potential weapon in our arsenalto combat antimicrobial resistance (AMR). However, there are key biologicaland physical questions remaining that need to be addressed to enhance thedevelopment of LTP for use in society. For example, the physical mechanismwhereby the reactive species in LTP damage the bacterial envelope and rendera cell non-viable is very poorly understood (e.g. Are pores formed in themembrane?). To further develop and optimise the use of LTP, a greaterunderstanding of the cellular mechanisms of its bactericidal effects is required,and this will be the focus of this project.Our current understanding of how LTP affects microbes is largely based onpopulation-level cell viability assays or single-cell imaging techniques with poortemporal resolution due to the need for extensive sample preparation prior toimaging. By combining our expertise in real-time single-cell fluorescenceimaging (CGB), LTP generation (DO'C), and bacterial phenotyping acrossdifferent time and length scales (MvdW), significant strides forward can bemade in understanding how plasma treatment alters the membrane bilayerboth in vivo and in vitro. This knowledge will feed into ongoing work in the YorkPlasma Institute that focuses on characterising and manipulating thecomposition of the reactive species in plasma to enhance biological activity.This project will investigate how different LTPs affect the cell envelope of Gramnegative bacterial strains with different cell surfaces by using a single-cell levelapproach. The student will exploit methods for fluorescently-labelling OMPsand LPS to monitor morphological changes in the cell envelope during and afterexposure to LTP. Single-cell imaging will be done using advanced multi-colourfluorescence microscopy methods and novel technology developed to allowsimultaneous exposure to different types and dosages of LTP. Time-lapse cellimaging after exposure to LTP will be done to determine if any morphologicaldifferences can be observed, and to identify potential phenotypic traits of non-viable cells and viable persister cells. Knowledge gained from this project will beused to develop a mechanistic model of LTP-induced membrane damage andidentify potential agents that could be used to enhance the damaging effect ofplasma on Gram negative bacteria.This project will provide excellent specialised training in cutting-edge single-cellanalytical methods combined with a good understanding of membrane biologyand microbiology. Professional skills training will be provided by the White RoseDTP. The student will also improve their professional skills by attending thesupervisors' lab meetings, departmental seminars and research conferences,and participating in outreach activities. The student will join a vibrant cross-disciplinary community of PhD students.This project is suitable for an applicant with a strong background in physics andchemistry, and a keen interest in understanding biological processes at themolecular level and participating in the development of novel antibacterialapproaches.