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

全球挑战包括微生物感染、表面（如食物）和环境的细菌污染。随着对抗生素耐药性的日益关注，需要开发替代方法。其中之一是低温等离子体（LTP）。当对气流施加高压时，形成具有生物活性的活性氧和氮气的混合物。常压LTP的化学性质为对抗伤口和皮肤溃疡相关的局部细菌感染提供了无抗生素治疗的巨大希望，是我们对抗抗菌素耐药性（AMR）的另一种潜在武器。然而，为了促进LTP在社会中的应用，仍有一些关键的生物学和物理学问题需要解决。例如，LTP中的活性物质破坏细菌包膜并使细胞无法存活的物理机制尚不清楚（例如，膜上是否形成了孔？）为了进一步开发和优化LTP的使用，需要对其杀菌作用的细胞机制有更深入的了解，这将是本项目的重点。我们目前对LTP如何影响微生物的理解主要基于群体水平的细胞活力测定或单细胞成像技术，由于需要在成像前进行大量的样品制备，这些技术的时间分辨率很低。通过结合我们在实时单细胞荧光成像（CGB）、LTP生成（DO'C）和不同时间和长度尺度（MvdW）的细菌表型方面的专业知识，可以在了解血浆处理如何改变体内和体外膜双层方面取得重大进展。这些知识将用于约克等离子体研究所正在进行的工作，重点是表征和操纵等离子体中活性物质的组成，以增强生物活性。本项目将通过单细胞水平的方法研究不同LTPs如何影响具有不同细胞表面的革兰氏阴性菌株的细胞包膜。该学生将利用荧光标记omp和LPS的方法来监测暴露于LTP期间和之后细胞包膜的形态学变化。单细胞成像将使用先进的多色荧光显微镜方法和新技术进行，以允许同时暴露于不同类型和剂量的LTP。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学差异，并确定非活细胞和活的持久性细胞的潜在表型特征。从该项目中获得的知识将用于开发ltp诱导膜损伤的机制模型，并确定可用于增强血浆对革兰氏阴性菌损伤作用的潜在药物。该项目将提供先进的单细胞分析方法的优秀专业培训，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White RoseDTP提供。学生还将通过参加导师的实验室会议、部门研讨会和研究会议以及参加外展活动来提高他们的专业技能。该学生将加入一个充满活力的跨学科博士生社区。本项目适合具有较强的物理和化学背景，对分子水平上的生物过程有浓厚的兴趣，并参与新型抗菌方法的开发。

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.