Characterization of iron regulon in Bacillus anthracis

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，两者都编码在大质粒上。 吸入性炭疽是该疾病最严重的形式，是一种全身性感染，其中生物体扩散到淋巴结，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。 染色体上编码的因子也与毒力有关，因为某些缺乏毒力质粒的菌株仍然能够在免疫动物中引起疾病。这些“疫苗耐药”菌株作为生物恐怖主义武器的未来发展是主要关注的问题，并强调建立一个基本的了解，在这种细菌的发病机制的分子机制的重要性。 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需元素铁。 铁的获取所涉及的机制已被证明对于许多细菌病原体的毒力是重要的，包括在血液中复制的生物体，其中大部分可用的铁被宿主铁化合物如转铁蛋白和血红蛋白（红细胞中）螯合。 没有研究检查B.炭疽菌在体内或体外生长期间获得铁。 这是一个尚未探索的重要研究领域，在这项拟议的研究中，我打算测试的假设，参与运输和利用宿主铁化合物的毒力因子存在于B。炭疽病 我们的长期兴趣是确定参与利用宿主铁化合物的表面蛋白，并确定这些蛋白质是否可能是有用的疫苗候选物。 我们开发了一种低铁基本培养基来培养B。炭疽杆菌，并使用这种培养基显示细菌可以使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。 在过去的一年中，我们独立开发了一种在B染色体中构建定点突变的方法。炭疽病 该技术已用于构建参与B生物合成的基因突变。炭疽菌含铁细胞 铁载体突变导致产生铁螯合活性降低的菌株，但仍然产生可检测量的细胞外铁螯合化合物。 这些结果表明，B。炭疽菌可产生多种铁载体。 在试图确定参与利用转铁蛋白和乳铁蛋白作为铁源的蛋白质，我们已经使用亲和色谱富集铁调节膜蛋白结合转铁蛋白和乳铁蛋白。 几种蛋白质，有能力结合转铁蛋白和乳铁蛋白部分纯化使用这种技术和它们的N-末端的氨基酸序列目前正在确定。 从这些蛋白质获得的N-末端序列将用于鉴定编码这些不同蛋白质的基因。 基因及其相关产品，似乎是直接参与利用转铁蛋白或乳铁蛋白作为铁源将进行更详细的检查。

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.