Search for pathogenic factor in Rickettsia tsutsupamushi.

恙虫病立克次体存在多种抗原变异。这种抗原性变体是由于位于立克次体表面的56千道尔顿（K）蛋白的抗原性。近年来我们发现，立克次体的抗原类型与其对小鼠的毒力有关。提示56 K蛋白可能决定了该立克次体的毒力。因此，本研究采用生物化学和基因克隆的方法，对Gilliam、Karp和Kato 3个强毒株和Shimokoshi、川崎和Kuroki 3个弱毒株的56 K蛋白进行了分子结构分析，比较了不同毒株之间的分子结构差异。所得结果总结如下。（1）在克隆基因的5 '端侧翼区推导出启动子序列和核糖体结合螨（ribosomebindingMite）序列，其序列分别与大肠杆菌的共有序列和Shine-Dalgarno序列相似。（2）6株菌推导的氨基酸序列的N端均含有由22个氨基酸组成的信号肽。(3)In鉴定出56 K蛋白分子，4个可变区，氨基酸序列在菌株间存在显著差异。（4）疏水区和亲水区交替出现，具有典型的膜蛋白性质。4个可变区均位于亲水区，提示这些可变区暴露在立克次体表面，可能存在抗原位点（表位）。(5)A除川崎株外，在终止密码子下游约20 bp处的基因3 '侧侧翼区观察到发夹环结构。（6）不同菌株间氨基酸序列的变异比预期的要大、复杂。为了确定与立克次体毒力相关的序列，需要对该蛋白进行更详细的研究。

There are several antigenic variants in Rickettsia tsutsugamushi. This antigenic variant is due to the antigenicities of 56-kilodalton(K)protein located on the rickettsial surface. We found recently that the virulence of individual strain to mice relates to the antigenic type of rickettsiae. This result suggests that the 56K protein may decide the virulence of this rickettsia. Therefore we analyzed, in this study, the molecular structure of 56K proteins from 3 virulent strains of Gilliam, Karp, and Kato, and 3 avirulent strains of Shimokoshi, Kawasaki, and Kuroki, by biochemical and gene-cloning methods, and compared the differences of the molecular structure among the strains. The results obtained are summarized as follows. (1)Promoter sequences and ribosome-binding Mite were deduced in the 5'-side flanking regions of cloned genes, of which the sequences are similar with the consensus and Shine-Dalgarno sequences in Escherichia coli, respectively. (2)Signal peptide composed of 22-amino acids was observed at the N-terminal side of the deduced amino acid sequences from all the six strains. (3)In the 56K protein molecules, 4 variable domains showing remarkable variations of amino acid sequences among the strains were recognized. (4)Hydrophobic and hydrophilic regions appeared alternatively in the molecules, showing typical properties of membrane protein. All the 4 variable domains located in the hydrophilic regions, suggesting that these domains are exposed on the surface of rickettsiae and antigenic site(epitope)may exist in these areas. (5)A hairpin loop structure was seen at the 3'-side flanking region of the gene about 20 bp downstream from the stop codon except the case of Kawasaki strain. (6)The variations of amino acid sequences among the strains were larger and more complex than we expected. To identify the sequence which relates to the virulence of the rickettsia, more detail studies on this protein are required in future.