Hammamieh Rasha, Ribot Wilson J, Abshire Terry G, Jett Marti, Ezzell John
Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
BMC Infect Dis. 2008 Sep 22;8:124. doi: 10.1186/1471-2334-8-124.
Anthrax is caused by Bacillus anthracis that produce two exotoxins, lethal toxin and edema toxin. The lethal toxin is composed of the lethal factor (LF) complexed with the cell binding protective antigen (PA83, 83 kDa). Likewise, the edema factor (EF) binds to the PA83 to form the edema toxin. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the full-length, inactive protein into 63 kDa and 20 kDa antigens (PA63 and PA20). PA63 forms a heptamer and is internalized via receptor mediated endocytosis forming a protease-stable pore, which allows EF and LF to enter the cell and exert their toxic effects.Both proteolytically cleaved protective antigens (PA63 and PA20 fragments) are found in the blood of infected animals. The 63 kDa protective antigen PA63 fragment has been thoroughly studied while little is known about the PA20.
In this study we examined the role of PA20 using high throughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to the PA20. We constructed a PA mutant in which a Factor Xa proteolytic recognition site was genetically engineered into the protective antigen PA83 to obtain PA20 using limited digestion of this recombinant PA83 with trypsin.
Global gene expression response studies indicated modulation of various immune functions and showed gene patterns indicative of apoptosis via the Fas pathway in a subset of the lymphoid cells. This finding was extended to include observations of increased Caspase-3 enzymatic activity and the identification of increases in the population of apoptotic, but not necrotic cells, based on differential staining methods. We identified a list of approximately 40 inflammatory mediators and heat-shock proteins that were altered similarly upon exposure of PBMC to either rPA20 or B. anthracis spores/vegetative cells.
This study shows that the PA20 has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.
炭疽由炭疽杆菌引起，该菌可产生两种外毒素，即致死毒素和水肿毒素。致死毒素由与细胞结合保护性抗原（PA83，83 kDa）复合的致死因子（LF）组成。同样，水肿因子（EF）与PA83结合形成水肿毒素。一旦PA83与宿主细胞表面结合，一种类弗林蛋白酶将全长无活性蛋白切割成63 kDa和20 kDa抗原（PA63和PA20）。PA63形成七聚体，并通过受体介导的内吞作用内化，形成蛋白酶稳定的孔道，使EF和LF能够进入细胞并发挥其毒性作用。在受感染动物的血液中均发现了经蛋白水解切割的保护性抗原（PA63和PA20片段）。63 kDa保护性抗原PA63片段已得到充分研究，而关于PA20的了解却很少。
在本研究中，我们通过对暴露于PA20的人外周血单个核细胞（PBMC）进行高通量基因表达分析，研究了PA20的作用。我们构建了一个PA突变体，其中在保护性抗原PA83中通过基因工程引入了一个凝血因子Xa蛋白水解识别位点，通过用胰蛋白酶对该重组PA83进行有限消化来获得PA20。
整体基因表达反应研究表明，多种免疫功能受到调节，并显示出在一部分淋巴细胞中通过Fas途径指示细胞凋亡的基因模式。这一发现进一步扩展，包括观察到半胱天冬酶-3酶活性增加，以及基于差异染色方法鉴定出凋亡细胞群体增加，而非坏死细胞群体增加。我们确定了一份约40种炎症介质和热休克蛋白的清单，当PBMC暴露于rPA20或炭疽芽孢杆菌孢子/营养细胞时，它们的变化情况相似。
本研究表明，PA20对人外周血白细胞有影响，并且在没有其他PA成分的情况下可诱导细胞凋亡。
