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

金黄色葡萄球菌是一种主要的、对抗生素具有耐药性的人类病原体。针对细菌细胞壁，通过抗生素(如β-内酰胺类)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，并参与休克、关节炎等。尽管如此，由于获得足够材料的技术困难，我们不知道PG在感染过程中的结构。这项跨学科的项目将跨越从单分子到整个动物的长度范围，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期间的第一个PG结构，以及特定的体内产生的结构在疾病中的作用。科学目的：1.体外生产和分析肽聚糖结构金黄色葡萄球菌PG的结构将使用在富含介质和模拟体内条件(全人血液)的体外培养的细菌来确定。反相高效液相色谱分离，结合MS和核磁共振，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。将使用谢菲尔德开发的一种协议来测量葡聚糖链长度。原子力显微镜将决定PG的结构。对检测灵敏度的分析将确定所需的细菌数量(对体内分析很重要)。2.分析肽聚糖修饰在宿主中的作用：病原体相互作用学生将使用我们建立的成熟的小鼠模型，接受体内金黄色葡萄球菌感染分析的培训。这需要内政部的个人执照。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是在体内生长的，因为每个细菌都是由一种单独的细菌建立的。脓肿可以包含多达109个CFU，初步数据表明这足以进行分析。已经建立了一种从脓肿中获取细菌的方案。将PG进行纯化，并将其结构和构筑与体外衍生材料进行比较。3.分析PG修饰在宿主中的作用：病原体的相互作用。体内相关细胞壁特征的重要性将通过使用特定的突变体来检验(已有PG水解酶、O-乙酰转移酶等基因的金黄色葡萄球菌突变体的有序文库)。已知O-乙酰化等修饰在宿主-病原菌相互作用中起着重要作用。PG结构具有多种功能，将被测试，包括通过酶抵抗宿主攻击和脱落物质对免疫系统的影响(细胞因子分析等)。耐甲氧西林金黄色葡萄球菌(MRSA)是一个主要的卫生问题。甲氧西林和其他β-内酰胺类药物的靶标是PG的生物合成。MRSA PG的体内结构以及抗生素是如何影响的尚不清楚。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模式如何阻止有效的控制。

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.