The role of S.aureus cell wall structure during host:pathogen interaction

金黄色葡萄球菌细胞壁结构在宿主与病原体相互作用中的作用

• 批准号: 1812138
• 金额: --
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1812138
• 关键词: role; S.aureus; cell; wall; structure

S. aureus is a major, antibiotic resistant, human pathogen. Targeting bacterial cell wall, peptidoglycan (PG) biosynthesis via antibiotics such as the beta-lactams has been a crucial part in our fight against S. aureus. PG is also recognized by the host, and is involved in shock, arthritis etc. Despite this importance we do not know the structure of the PG during infection, hampered by technological difficulties in obtaining enough material. The interdisciplinary project, in infectious disease will span the length scales from single molecules to whole animals, combining host:pathogen interaction, molecular imaging and biochemistry to define the first PG structure during infection and the role of specific in vivo produced structures in disease. Scientific Objectives:1. Production and analysis of peptidoglycan structure in vitroThe structure of S. aureus PG will be determined using bacteria grown in vitro in rich media and under conditions that mimic in vivo conditions (whole human blood). RP-HPLC separation of muropeptides, coupled with MS and NMR will define the structure and modification of S. aureus PG (Newcastle and Sheffield). Glycan strand length will be measured using a protocol developed in Sheffield. Atomic force microscopy will determine PG architecture. Analysis of the sensitivity of the assays will determine the number of bacteria required (important for the in vivo analysis). 2. Analysis of the role of modification of peptidoglycan during host:pathogen interactionThe student will be trained in in vivo analysis of S. aureus infection, using our well-established mouse models. This requires a Home Office personal licence. The student will then use the mouse sepsis model, a primary bacterial output of the model being abscesses in organs. All the abscess-associated bacteria have grown in vivo as each is founded by an individual bacterium. Abscesses can contain up to 109 cfu, which preliminary data suggests is enough PG for analysis. A protocol has already been established for harvesting bacteria from abscesses. PG will be purified and its structure and architecture compared to in vitro derived material. 3. Analysis of the role of PG modifications in host:pathogen interaction.The importance of in vivo associated cell wall features will be tested by the use of specific mutants (an ordered library of S. aureus mutants in genes such as PG hydrolases, O-acetyltransferase etc.is available). It is known that modifications such as O-acetylation have an important role in host:pathogen interaction. PG structure has multiple functions that will be tested including resistance to host attack via enzymes and the effect of shed material on the immune system (cytokine analysis etc). 4. Role of antibiotic resistance and treatment on PG in vivo structureMethicillin Resistant S. aureus (MRSA) is a major healthcare problem. The target of methicillin and other beta-lactams is PG biosynthesis. The in vivo structure of MRSA PG and how antibiotics effect this is unknown. If we are to identify new treatment regimes it is important to determine how existing resistance modalities prevent effective control.

金黄色葡萄球菌是一种主要的、具有抗生素耐药性的人类病原体。针对细菌细胞壁，通过β-内酰胺等抗生素生物合成肽聚糖（PG）一直是我们对抗金黄色葡萄球菌的关键部分。 PG也被宿主识别，并与休克、关节炎等有关。尽管如此重要，但由于获得足够材料的技术困难，我们仍不知道感染期间PG的结构。这个传染病领域的跨学科项目将跨越从单分子到整个动物的长度尺度，结合宿主：病原体相互作用、分子成像和生物化学来定义感染过程中的第一个PG结构以及特定体内产生的结构在疾病中的作用。科学目标： 1．体外肽聚糖结构的产生和分析金黄色葡萄球菌PG的结构将使用在丰富培养基中和在模拟体内条件（全人血）的条件下体外生长的细菌来确定。室肽的 RP-HPLC 分离，结合 MS 和 NMR 将确定金黄色葡萄球菌 PG（纽卡斯尔和谢菲尔德）的结构和修饰。聚糖链长度将使用谢菲尔德开发的协议进行测量。原子力显微镜将确定 PG 结构。测定灵敏度的分析将确定所需的细菌数量（对于体内分析很重要）。 2. 分析肽聚糖修饰在宿主与病原体相互作用过程中的作用学生将接受使用我们完善的小鼠模型对金黄色葡萄球菌感染进行体内分析的培训。这需要内政部个人许可证。然后，学生将使用小鼠败血症模型，该模型的主要细菌输出是器官中的脓肿。所有与脓肿相关的细菌都在体内生长，因为每种细菌都是由单个细菌产生的。脓肿可含有高达 109 cfu，初步数据表明这足以进行分析。已经制定了从脓肿中收获细菌的方案。 PG 将被纯化，并将其结构和架构与体外衍生材料进行比较。 3.分析PG修饰在宿主：病原体相互作用中的作用。体内相关细胞壁特征的重要性将通过使用特定突变体进行测试（可以使用PG水解酶、O-乙酰转移酶等基因中的金黄色葡萄球菌突变体的有序文库）。众所周知，O-乙酰化等修饰在宿主：病原体相互作用中具有重要作用。 PG 结构具有多种将被测试的功能，包括通过酶抵抗宿主攻击以及脱落物质对免疫系统的影响（细胞因子分析等）。 4. 抗生素耐药性和治疗对 PG 体内结构的作用耐甲氧西林金黄色葡萄球菌 (MRSA) 是一个主要的医疗保健问题。甲氧西林和其他 β-内酰胺类药物的目标是 PG 生物合成。 MRSA PG 的体内结构以及抗生素如何影响它尚不清楚。如果我们要确定新的治疗方案，重要的是要确定现有的耐药方式如何阻碍有效控制。