Characterization of iron regulon in Bacillus anthracis

炭疽杆菌中铁调节子的表征

• 批准号: 6678347
• 负责人: MICHAEL P SCHMITT
• 金额: --
• 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/6678347
• 关键词: Bacillus anthracis; affinity chromatography; anthrax; bacteria infection mechanism; bacterial genetics; bacterial proteins; biological transport; bioterrorism /chemical warfare; ferritin; gene mutation; genetic regulatory element; heme; hemoglobin; iron; lactoferrin; siderophores; site directed mutagenesis; transferrin; vaccine development; virulence

Summary: Anthrax is a severe disease caused by the gram-positive bacterium Bacillus anthracis. The organism infects humans and many other animals and the only identified virulence factors are anthrax toxin and a glutamic acid capsule, both of which are encoded on large plasmids. Inhalation anthrax, the most severe form of the disease, is a systemic infection in which the organism spreads to the lymph nodes and then into the blood where it is able to replicate to levels as high as 100 million bacteria / ml. Factors encoded on the chromosome have also been implicated in virulence, since certain strains lacking the virulence plasmids are still capable of causing disease in immunized animals. The future development of these "vaccine resistant" strains as bioterrorism weapons is of major concern, and stresses the importance of establishing a fundamental understanding of the molecular mechanisms involved in the pathogenesis of this bacterium. For many bacterial pathogens, the ability to survive in the human host requires the acquisition of the essential element iron. Mechanisms involved in the acquisition of iron have been shown to be important for the virulence of numerous bacterial pathogens, including organisms that replicate in the blood, where much of the available iron is sequestered by host iron compounds such as transferrin and hemoglobin (in erythrocytes). No investigations have examined the mechanism by which B. anthracis acquires iron during growth in vivo or in vitro. This is an important area of study that has not been explored, and in this proposed research, I intend to test the hypothesis that virulence factors involved in the transport and utilization of host iron compounds are present in B. anthracis. Our long-term interest is to identify surface proteins that are involved in the utilization of host iron compounds, and determine if these proteins may be useful vaccine candidates. We have developed a low-iron minimal medium to cultivate B. anthracis and have used this medium to show that the bacterium could use a variety of host compounds as essential iron sources, including heme, hemoglobin, ferritin, transferrin, and lactoferrin. In the past year we have independently developed a method for constructing site directed mutations in the chromosome of B. anthracis. This technique has been used to construct mutations in genes involved in the biosynthesis of the B. anthracis siderophore. The siderophore mutations resulted in strains that had diminished production of iron chelating activity, but nevertheless still made detectable quantities of an extracellular iron-chelating compound. These findings suggested that B. anthracis may produce multiple siderophores. In an attempt to identify proteins involved in the utilization of transferrin and lactoferrin as iron sources, we have used affinity chromatography to enrich for iron-regulated membrane proteins that bind to transferrin and lactoferrin. Several proteins that have the ability to bind to transferrin and lactoferrin were partially purified using this technique and the amino acid sequence of their N-terminal is currently being determined. The N-terminal sequence obtained from these proteins will be used to identify the genes that encode these various proteins. Genes and their associated products that appear to be directly involved in the utilization of either transferrin or lactoferrin as iron sources will be examined in greater detail.

摘要：炭疽病是由革兰氏阳性细菌炭疽杆菌引起的一种严重疾病。这种微生物感染人类和许多其他动物，唯一确定的毒力因子是炭疽毒素和谷氨酸胶囊，这两种毒素都编码在大质粒上。吸入性炭疽是该病最严重的形式，是一种全身性感染，在这种感染中，微生物会传播到淋巴结，然后进入血液，在那里它能够复制到高达1亿个细菌/毫升的水平。染色体上编码的因素也与毒力有关，因为某些缺乏毒力质粒的菌株仍然能够在免疫动物中致病。这些“疫苗耐药”菌株作为生物恐怖主义武器的未来发展令人关注，并强调了对这种细菌致病的分子机制建立基本了解的重要性。 对于许多细菌病原体来说，在人类宿主中生存的能力需要获得必要的元素铁。铁的获取机制已被证明对许多细菌病原体的毒力非常重要，包括在血液中复制的生物体，其中大部分可用铁被宿主铁化合物隔离，如转铁蛋白和血红蛋白(在红细胞中)。还没有研究检验炭疽杆菌在体内或体外生长过程中获得铁的机制。这是一个尚未被探索的重要研究领域，在这项拟议的研究中，我打算检验一种假设，即炭疽杆菌中存在参与宿主铁化合物运输和利用的毒力因子。 我们的长期兴趣是确定与宿主铁化合物的利用有关的表面蛋白，并确定这些蛋白是否可能是有用的候选疫苗。我们开发了一种低铁最低限度的培养炭疽杆菌的培养基，并用该培养基表明该细菌可以利用多种宿主化合物作为必需的铁源，包括血红素、血红蛋白、铁蛋白、转铁蛋白和乳铁蛋白。在过去的一年里，我们自主开发了一种在炭疽杆菌染色体上构建定点突变的方法。这项技术已被用于构建与炭疽杆菌铁载体生物合成有关的基因突变。铁载体的突变导致菌株减少了铁螯合活性的产生，但仍然产生了可检测到的细胞外铁螯合化合物。这些发现表明，炭疽杆菌可能产生多个铁载体。为了鉴定转铁蛋白和乳铁蛋白作为铁源的相关蛋白质，我们利用亲和层析法对铁调节的膜蛋白进行了富集化，这些蛋白与转铁蛋白和乳铁蛋白结合。用这种技术部分纯化了几种能够与转铁蛋白和乳铁蛋白结合的蛋白质，目前正在测定它们的N末端的氨基酸序列。从这些蛋白质中获得的N末端序列将用于识别编码这些不同蛋白质的基因。将更详细地研究似乎直接涉及转铁蛋白或乳铁蛋白作为铁源利用的基因及其相关产物。