REGULATIONS OF IRON ACQUISITATION BY S AUREUS

S AUREUS 获取铁的规定

• 批准号: 6091811
• 负责人: RADHESHYAM K JAYASWAL
• 金额: $ 13.01万
• 依托单位: ILLINOIS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6091811
• 关键词: DNA footprinting; Staphylococcus aureus; bacterial genetics; disease /disorder model; endocarditis; gel mobility shift assay; gene mutation; iron metabolism; laboratory rat; microorganism metabolism; molecular cloning; northern blottings; operon; site directed mutagenesis; virulence

DESCRIPTION (Adapted from the Applicant's Abstract): Staphylococcus aureus is a major cause of human diseases, ranging from basic skin abscesses to complicated life-threatening diseases such as toxic shock syndrome, pneumonia, septicemia, and infective endocarditis. The acquisition of broad antibiotic resistance in this bacterium, especially the methicillin and vancomycin-resistant S. aureus strains, poses a major threat to public health. The virulence factors involved in the pathogenicity of S. aureus have been extensively studied, but the molecular mechanisms governing S. aureus invasion, colonization, and survival within the host environment remains largely unknown. Various stress conditions within the host are proposed to simulate the expression of virulence genes in S. aureus. Iron, which has been shown to be an important virulence determinant and required for many vital cellular processes, is not readily available for bacterial cells in the animal host. To cope with an iron-deficient host environment, the bacteria have evolved several mechanisms of iron acquisition and its regulation. However, these pathways are poorly understood in S. aureus. The PI has recently cloned and partially characterized a S. aureus chromosomal gene, fur, that shows homology to the ferric-uptake regulatory genes of other bacteria. The fur gene codes for a protein that binds to a specific DNA sequence in the promoter region of at least two genes, sirA and fhu. The long-term goal of this project is to investigate how Fur regulates iron acquisition in S. aureus and its role in the regulation of the expression of virulence genes. The specific objectives of this study are 1) cloning and molecular analysis of fur, 2) construction of a mutation in fur and determination of its effect on pathogenicity, 3) molecular analysis of Fur, and 4) characterization of the Fur regulated iron uptake operon, fhu of S. aureus. To accomplish this first objective, the PI has cloned fur which will be overexpressed and the protein purified to conduct gel-shift and footprint assays. Northern blots will be done to determine the mechanism of fur regulation. To accomplish the second objective, Fur- mutants will be generated by site-directed mutagenesis and the effect of the mutation on pathogenicity will be determined using a rat model of endocarditis. The Fur- mutant and the parent will be used to identify Fur-regulated genes in an in vitro cycle selection procedure or alternatively by 2-D gel electrophoresis to accomplish the third objective. The fourth objective will be accomplished by sequencing the fhu operon, followed by the construction of a mutation in each reading frame to determine their roles in the pathogenicity and uptake of iron. The results of this study may identify novel targets that interfere with iron uptake and its regulation and may lead to alternative therapies for staphylococcal infections.

描述（根据申请人的摘要改编）：金黄色葡萄球菌是 人类疾病的主要原因，从基本的皮肤脓肿到 复杂的威胁生命的疾病，例如有毒休克综合征，肺炎， 败血病和感染性心内膜炎。 获取广泛的抗生素 该细菌的耐药性，尤其是甲氧西林和 抗Vanomycin的金黄色葡萄球菌菌株对公共卫生构成了重大威胁。 金黄色葡萄球菌致病性涉及的毒力因子已经 经过广泛的研究，但分子机制管理金黄色葡萄球菌入侵， 殖民化和宿主环境中的生存在很大程度上仍然存在 未知。 提出了主机内的各种应力条件以模拟 金黄色葡萄球菌中毒力基因的表达。 铁，已被证明 成为许多重要细胞的重要毒力决定因素，并且需要 在动物宿主中的细菌细胞不容易获得过程。 到 应对缺铁的宿主环境，细菌已经进化了几种 铁的机制及其调节。 但是，这些途径是 在金黄色葡萄球菌中鲜为人知。 PI最近克隆并部分表征了金黄色葡萄球菌染色体 基因，毛皮，表明与其他其他其他摄取调节基因同源 细菌。 皮草基因代码与特定DNA结合的蛋白质代码 至少两个基因Sira和Fhu的启动子区域中的序列。 这 该项目的长期目标是研究毛皮如何调节铁 金黄色葡萄球菌的获取及其在调节表达中的作用 毒力基因。 这项研究的具体目标是1）克隆和 毛皮的分子分析，2）在毛皮和 确定其对致病性的影响，3）毛发的分子分析， 4）表征毛皮调节的铁摄取操纵子，金黄色葡萄球菌的FHU。 为了实现这个第一个目标，PI克隆了皮毛，这将是 过表达，蛋白质纯化以进行凝胶换挡和足迹 测定。 将进行北印迹以确定皮毛的机制 规定。 为了实现第二个目标，将产生毛发突变体 通过定点诱变和突变对致病性的影响 将使用心内膜炎大鼠模型确定。 毛皮和 父会在体外循环中识别受毛管调节的基因 选择程序或通过二维凝胶电泳进行选择 第三个目标。 第四个目标将通过排序来实现 FHU操纵子，然后在每次阅读中构建突变 框架确定其在铁的致病性和摄取中的作用。 这 这项研究的结果可能会确定干扰铁的新型目标 吸收及其法规，可能会导致替代疗法 葡萄球菌感染。

项目成果

Fur is required for the activation of virulence gene expression through the induction of the sae regulatory system in Staphylococcus aureus.

• DOI: 10.1016/j.ijmm.2010.05.003
• 发表时间: 2011-01
• 期刊: INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY
• 影响因子: 4.1
• 作者: Johnson, Miranda;Sengupta, Mrittika;Purves, Joanne;Tarrant, Emma;Williams, Peter H.;Cockayne, Alan;Muthaiyan, Arunachalam;Stephenson, Robert;Ledala, Nagender;Wilkinson, Brian J.;Jayaswal, Radheshyam K.;Morrissey, Julie A.
• 通讯作者: Morrissey, Julie A.