Mukhopadhyay Sanghamitra, Akmal Arya, Stewart Andrew C, Hsia Ru-Ching, Read Timothy D
Biological Defense Research Directorate, Naval Medical Research Center, Rockville, Maryland 20852, USA.
Mol Cell Proteomics. 2009 Jun;8(6):1174-91. doi: 10.1074/mcp.M800403-MCP200. Epub 2009 Feb 9.
We sought to identify proteins in the Bacillus anthracis spore, conserved in other strains of the closely related Bacillus cereus group, that elicit an immune response in mammals. Two high throughput approaches were used. First, an in silico screening identified 200 conserved putative B. anthracis spore components. A total of 192 of those candidate genes were expressed and purified in vitro, 75 of which reacted with the rabbit immune sera generated against B. anthracis spores. The second approach was to screen for cross-reacting antigens in the spore proteome of 10 diverse B. cereus group strains. Two-dimensional electrophoresis resolved more than 200 protein spots in each spore preparation. About 72% of the protein spots were found in all the strains. 18 of these conserved proteins reacted against anti-B. anthracis spore rabbit immune sera, two of which (alanine racemase, Dal-1 and the methionine transporter, MetN) overlapped the set of proteins identified using the in silico screen. A conserved repeat domain protein (Crd) was the most immunoreactive protein found broadly across B. cereus sensu lato strains. We have established an approach for finding conserved targets across a species using population genomics and proteomics. The results of these screens suggest the possibility of a multiepitope antigen for broad host range diagnostics or therapeutics against Bacillus spore infection.
我们试图在炭疽芽孢杆菌孢子中鉴定出在密切相关的蜡样芽孢杆菌群的其他菌株中保守的、能在哺乳动物中引发免疫反应的蛋白质。我们采用了两种高通量方法。首先，通过计算机筛选确定了200个保守的假定炭疽芽孢杆菌孢子成分。其中共有192个候选基因在体外表达并纯化，其中75个与针对炭疽芽孢杆菌孢子产生的兔免疫血清发生反应。第二种方法是在10种不同的蜡样芽孢杆菌群菌株的孢子蛋白质组中筛选交叉反应抗原。二维电泳在每种孢子制剂中分辨出200多个蛋白质斑点。约72%的蛋白质斑点在所有菌株中都能找到。其中18种保守蛋白质与抗炭疽芽孢杆菌孢子兔免疫血清发生反应，其中两种（丙氨酸消旋酶Dal-1和甲硫氨酸转运蛋白MetN）与通过计算机筛选鉴定出的蛋白质组重叠。一种保守重复结构域蛋白（Crd）是在广泛的蜡样芽孢杆菌属菌株中发现的免疫反应性最强的蛋白质。我们已经建立了一种利用群体基因组学和蛋白质组学在一个物种中寻找保守靶点的方法。这些筛选结果表明，有可能开发出一种多表位抗原，用于针对芽孢杆菌孢子感染的广泛宿主范围诊断或治疗。
