Guina T, Oliver D B
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Mol Microbiol. 1997 Jun;24(6):1201-13. doi: 10.1046/j.1365-2958.1997.4291786.x.
We cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli. The haemolytic activity was erythrocyte-species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively. Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31. blyA encodes a predicted alpha-helical 7.4 kDa protein with a hydrophobic central region and a positively charged C-terminus, which is structurally homologous to a large group of pore-forming toxins with cytolytic activity. blyB encodes a soluble protein which stabilized BlyA and enhanced haemolytic activity. While the majority of BlyA in E. coli was membrane-associated, only soluble protein was haemolytically active. The haemolytic activity was shown to be highly protease sensitive, heat labile, independent of divalent cations, and extremely dependent on protein concentration, consistent with a requirement for oligomerization as the mechanism of action. BlyA was highly purified from E. coli in a single step utilizing Triton X-114 phase partitioning. Genetic analysis of blyA and blyB mutants indicated that the stability, membrane association, and activity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein. The bly genes were found to be expressed at a very low level in cultured B. burgdorferi.
我们通过伯氏疏螺旋体膜相互作用蛋白在大肠杆菌中的溶血活性克隆了其编码基因。该溶血活性具有红细胞物种特异性，对马、羊和兔的红细胞的活性依次递减。对溶血决定簇的遗传分析揭示了两个伯氏疏螺旋体溶血素基因，blyA和blyB，它们是一个预测的四基因操纵子的一部分，该操纵子在伯氏疏螺旋体B31的30 kb环形质粒上有多个拷贝。blyA编码一个预测的α螺旋7.4 kDa蛋白，其中心区域疏水，C端带正电荷，在结构上与一大类具有溶细胞活性的成孔毒素同源。blyB编码一种可溶性蛋白，可稳定BlyA并增强溶血活性。虽然大肠杆菌中的大多数BlyA与膜相关，但只有可溶性蛋白具有溶血活性。溶血活性显示对蛋白酶高度敏感、对热不稳定、不依赖二价阳离子且极其依赖蛋白质浓度，这与作为作用机制的寡聚化需求一致。利用Triton X-114相分配法在一步中从大肠杆菌中高度纯化了BlyA。对blyA和blyB突变体的遗传分析表明，BlyA的稳定性、膜结合和活性取决于其序列的细微变化以及BlyB蛋白。发现bly基因在培养的伯氏疏螺旋体中表达水平非常低。
