IRON AVAILABILITY IN THE PATHOGENESIS OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS

伴放线放线杆菌发病过程中铁的有效性

• 批准号: 3775558
• 负责人: Leslie J Winston
• 金额: --
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3775558
• 关键词: Actinobacillus actinomycetemcomitans; deficient growth media; genetic library; host organism interaction; ion exchange chromatography; iron metabolism; membrane proteins; microorganism culture; microorganism growth; molecular cloning; molecular pathology; nucleic acid probes; protein sequence; protein structure function

The role of Iron Availability in the Pathogenesis of Actinobacillus actinomycetemcomitans. The purpose of the project is to study the role of iron availability on the pathogenesis of Actinobacillus actinomycetemcomitans (A.a.). Little is known about the iron transport systems of anaerobic bacteria, although it is clear that the acquisition of iron from the host is required in order to sustain growth. The identification of an iron-repressible outer membrane protein of ~70 kilodaltons (kD) that is immunogenic in humans may represent a vaccine candidate for the prevention and treatment of A.a. infections. The specific aims of the study are to identify potential in vivo iron sources utilized by A.a to help in targeting iron transport systems. In addition, the role of the 70kD iron-repressible protein in iron uptake will be studied. To achieve these goals, an iron-limited growth medium will be developed in order to study iron utilization with a limited number of variables. We are currently employing 8-hydroxyquinoline extraction of complex media and cation exchange chromatography of defined media to produce an medium with ~100nM iron that will also support the growth of A.a. The gene for the 70kD protein will be cloned an sequenced to determine if it is similar to other know iron transport systems. An oligonucleotide probe derived form the N-terminal amino acid sequence of the 70kD protein was utilized to screen a ~gt11 library of A.a. genomic DNA to obtain potential clones. In addition, monospecific antisera generated against the 70kD antigen was also used for screening the library. Positive clones are currently being characterized to definitively demonstrate that we possess the gene for the 70kD protein. Once the wild-type gene has been identified, it will be subcloned into an appropriate vector and utilized to generate mutations in the wild- type gene in order to characterize the function of the protein in iron uptake. Keys Words: Actinobacillus, Iron, Outer Membrance Proteins, Iron Transport

铁的可用性在肌动杆菌发病机理中的作用 放线症。 该项目的目的是研究铁的作用 对静脉细菌的发病机理的可用性 放线菌（A.A.）。 对铁知之甚少 厌氧菌的运输系统，尽管很明显 需要从宿主那里获取铁才能 维持增长。 识别可抑制铁的外部 〜70千达尔顿（KD）的膜蛋白，该蛋白质是免疫原性的 人类可以代表预防疫苗的疫苗， A.A.的治疗感染。 该研究的具体目的是 确定A.A使用的体内铁源的潜力 在靶向铁运输系统中。 另外， 将研究70kD铁的铁蛋白摄取中的蛋白质。 到 实现这些目标，铁限制的生长媒介将是 为了研究有限数量的铁利用而开发 变量。 我们目前正在雇用8-羟基喹啉 提取复杂媒体和阳离子交换色谱 定义的培养基生产约100nm铁的培养基也将 支持A.A.的增长 70KD蛋白的基因将是 克隆一个测序以确定它是否类似于其他知道 铁运输系统。 寡核苷酸探针得出的形式 70KD蛋白的N末端氨基酸序列用于 屏幕A〜GT11库的A.A.基因组DNA获得电势 克隆。 此外，与 70kD抗原也用于筛选库。 积极的 克隆目前正在定义为明确的特征 证明我们拥有70KD蛋白的基因。 一旦 已经鉴定出野生型基因，将其子序列 适当的载体，并用于在野生中产生突变 键入基因以表征蛋白质的功能 铁吸收。 钥匙单词：肌动杆菌，铁，外膜蛋白，铁 运输