Search for pathogenic factor in Rickettsia tsutsupamushi.

恙虫病立克次体有几种抗原变异。这种抗原变异是由于位于立克次体表面的56千道尔顿(K)蛋白的抗原性。我们最近发现单个菌株对小鼠的毒力与立克次体的抗原类型有关。该结果提示56K蛋白可能决定了该立克次体的毒力。因此，本研究采用生化和基因克隆的方法分析了Gilliam、Karp、Kato 3株毒力菌株和Shimokoshi、Kawasaki、Kuroki 3株无毒菌株的56K蛋白的分子结构，并比较了菌株间分子结构的差异。所得结果总结如下：(1)在克隆基因的5′侧翼区推导出启动子序列和核糖体结合Mite序列，其序列分别与大肠杆菌的consensus和Shine-Dalgarno序列相似。(2) 6株菌株氨基酸序列的n端均有22个氨基酸组成的信号肽。(3)在56K蛋白分子中，识别出4个不同菌株间氨基酸序列差异显著的可变结构域。(4)疏水区和亲水区在分子中交替出现，表现出典型的膜蛋白特性。4个可变结构域均位于立克次体亲水性区域，表明这些结构域暴露在立克次体表面，抗原位点（表位）可能存在于这些区域。(5)除川崎菌株外，在停止密码子下游约20bp的3′侧侧翼区均可见发夹环结构。(6)菌株间氨基酸序列的差异比我们预期的更大、更复杂。为了确定与立克次体毒力有关的序列，未来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.