吉兰-巴雷综合征相关空肠弯曲菌cstⅡ基因序列对比研究

Comparison of cst Ⅱ gene in GuiHain-Barre syndrome-associated Campylobacter jejuni strains

目的研究华北地区3株吉兰-巴雷综合征（Guillain-Barre syndrome，GBS）相关空肠弯曲菌（Campylobacter jejuni，C.jejuni）的cstⅡ基因序列并寻找GBS相关及GBS无关的两类空肠弯曲菌菌株间可能与GBS致病性相关的基因突变位点和蛋白高级结构变化，分析菌株间遗传进化关系。方法选取分离自GBS患者粪便并经动物模型证实为致GBS的3株空肠弯曲菌菌株进行培养并提取基因组DNA测序，与GenBank中GBS无关空肠弯曲菌基因序列进行配对对比，寻找两类菌株间可能导致空肠弯曲菌致病能力差异的cstⅡ基因碱基突变位点及蛋白高级结构改变，观察这种变化是否为GBS相关菌株的共性，并构建遗传进化树。结果与GBS无关菌株相比，3株致GBS菌株cstⅡ基因的氨基酸序列二级结构的第7个0L螺旋的165—180区段均被打开形成了折叠或转角，该变化在其他GBS相关菌株的二级结构变化中同样得到体现。实验中75％的GBS相关菌株cstⅡ基因为双功能，25％GBS相关菌株及所有GBS无关菌株cstⅡ基因为单功能。LL株与实验中涉及的GBS相关菌株在进化上高度类聚。结论双功能cstⅡ可能与空肠弯曲菌的致GBS特性相关。LL株cstⅡ基因在一定程度上反映了亚洲地区的GBS相关菌株cstⅡ基因的序列特征，为进一步探索GBS发病的地域性特点并对GBS菌株进行监测提供了资料。

Objective To investigate the pathogenic mechanism of Campylobacter jejuni（ C. jejuni） associated with Guillain-Barr6 syndrome（GBS） and provide strategy for gene modification, the cst Ⅱ gene from 8 GBS-associated C. jejuni strains were compared with that from 3 GBS-unrelated C. jejuni strains, get- ting the base and amino acid mutations, the changes of secondary structures and finding the region which may be responsible for the pathogenicity of C. jejuni inducing GBS. Methods Three GBS-associated C. jeju- ni strains isolated from stools of GBS patients in north China were selected and cultured, which has been confirmed as GBS-associated by animal model. After sequencing the genome of them, the nucleotide se- quences of cst 11 gene were got through sequence alignment. The nucleotide sequences and deduced amini acid sequences of 3 GBS-associated cst Ⅱ genes were compared with that from 3 GBS-unrelated C. jejuni strains through bioinformatics software, getting the base and amino acid mutations, the changes of secondary structures. Other 5 GBS-associated cst Ⅱ genes were also aligned to know whether the differences we got above makes sense. In this way the genetic differences between two kinds of C. jejuni strains may be found and speculating the gene region related to the pathogenicity of GBS became possible. Results The cst Ⅱ gene of 3 GBS-associated C. jejuni strains were all composed of 876 base pairs. Compared with GBS-unrelat- ed C. jejuni strains, there were 9 consistent mutation sites in cst Ⅱ gene of LL and QYT stains, leading to 3 consistent amino acid mutation. The amino acid mutation of 114 and 182 sites in LL and QYT stains existed in other 5 GBS-associated C. jejuni strains. The sole amino acid mutation of ZHX strain -169 site, located near the 182 site. The seventh a-helix（ 165-180 region） of the secondary structure of the amino acid sequence from GBS-associated strains were shorter than that from GBS-unrelated strains, and the shorter re- gions were opened to form β-sheet or coli, which also existed in other GBS-related strains in this study. 75% of the GBS-associated cst Ⅱ genes were Asn-51, while 25% of the GBS-associated and all of the GBS-unrelated cst H genes were Thr-51. LL strain showed highly identity to other GBS-unrelated strains in this study. Conclusion The 165-180 segment of secondary structures in cst Ⅱgene from local 3 GBS-associated C. jeju- ni strains are probably the responsible region involved in inducing GBS. The senior structure changes in this region may affect the activity of sialyltransferase and the structures of ganglioside epitope, so that the C. jejuni can acquire the pathogenicity of GBS. This finding may give a clue to genetic modified site. The bi-function- al cst Ⅱ of C. jejuni may be related to the pathogenicity of GBS. The cst Ⅱ of LL strain to some extent repre- sents the characteristics of Asian strains, which may directs strains monitoring.