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Characterization of RTX toxins in Pasteurella pneumotropica and development of methods for detection and identification of the RTX toxins.

嗜肺巴氏杆菌中 RTX 毒素的表征以及 RTX 毒素检测和鉴定方法的开发。

基本信息

批准号：
20700369
负责人：
SASAKI Hiraku
金额：
$ 2.5万
依托单位：
Tokyo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

This study was conducted on identification of the proteins similar to RTX toxins and characterization of virulence properties of these toxins in rodent pathogen Pasteurella pneumotropica. Further, the methodology of detection and identification of these toxins was developed and evaluated. In genomic DNA of P. pneumotropica reference strain, total three proteins similar to RTX exoproteins-coding genes were identified. These RTX-like proteins were termed as PnxIA, PnxIIA, and PnxIIIA. All the recombinant proteins exhibited the cytotoxicity toward J774A.1 mouse macrophage cells. In particular, recombinant PnxIA and PnxIIA showed weak hemolytic activity toward sheep and mouse erythrocytes, otherwise recombinant PnxIIIA (rPnxIIIA) showed insignificant hemolytic activity. PnxIIIA has bacterial Ig-like domain and hemagglutinin repeat in primary structure. Thus, rPnxIIIA allowed to further evaluate extracellular matrices (ECMs) binding assay and hemagglutination assay. As results obtained from those experiments, rPnxIIIA showed significant binding ability toward rat collagen type I, and more than 30 ng/ml of rPnxIIIA showed hemagglutination of washed sheep erythrocytes. For the detection and identification of three RTX proteins in wild-type P. pneumotropica strains, genetic techniques and immunological methods were experimentally compared. However, these RTX proteins and -coding genes were highly diversified than those in reference strains. Therefore, it needs further evaluation and clarification of the molecular features of the RTX proteins in wild-type P. pneumotropica strains in future studies.

本研究对啮齿动物嗜肺巴氏杆菌RTX毒素的相似蛋白进行了鉴定，并对这些毒素的毒力特性进行了表征。此外，还开发和评价了检测和鉴定这些毒素的方法。在嗜肺假单胞菌参考菌株的基因组DNA中，鉴定了与RTX外蛋白编码基因相似的总共三种蛋白。这些RTX样蛋白被称为PnxIA、PnxIIA和PnxIIIA。所有重组蛋白对J774 A. 1小鼠巨噬细胞均有细胞毒活性。特别是，重组PnxIA和PnxIIA对绵羊和小鼠红细胞表现出弱的溶血活性，否则重组PnxIIIA（rPnxIIIA）表现出不显著的溶血活性。PnxIIIA在一级结构中具有细菌Ig样结构域和血凝素重复序列。因此，rPnxIIIA允许进一步评价细胞外基质（ECM）结合试验和血凝试验。从这些实验获得的结果是，rPnxIIIA显示出对大鼠I型胶原的显著结合能力，并且超过30 ng/ml的rPnxIIIA显示出洗涤的绵羊红细胞的血凝。为了检测和鉴定野生型嗜肺假单胞菌菌株中的三种RTX蛋白，对遗传技术和免疫学方法进行了实验比较。然而，这些RTX蛋白和编码基因比参考菌株中的那些高度多样化。因此，在未来的研究中，需要进一步评估和澄清野生型嗜肺假单胞菌菌株中RTX蛋白的分子特征。