Tn-seq法筛选高毒力肺炎克雷伯菌新毒力基因  

Screening of new virulence genes of high virulence Klebsiella pneumoniae by Tn-seq

作　　者：张婷 史继敏 陈甲龙 李刚 Zhang Ting;Shi Jimin;Chen Jialong;Li Gang(Department of Laboratory Medicine,Jinshan Hospital of Fudan University,Shanghai 201508,China)

机构地区：[1]复旦大学附属金山医院检验科,上海201508

出　　处：《中华微生物学和免疫学杂志》2021年第7期501-506,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的建立转座子突变文库,筛选高毒力肺炎克雷伯菌(hypervirulent Klebsiella pneumoniae,hvKp)的新毒力基因。方法构建转座子突变文库,血清处理,通过转座子测序(transposon sequencing,Tn-seq)比较分析血清处理前后各基因突变株在文库中丰度变化,并对筛选出的基因进行KEGG(kyoto encyclopedia of genes and genomes)注释和富集分析。结果以处理后丰度低于初始丰度20%为界,共筛选出405个基因,其中351个基因在HS11286、NJST258_1、NTUH-K2044和RJF293等4株参考菌株中保守存在,占86.7%,10个基因为NTUH-K2044和RJF293两株高毒力株共有而低毒力株HS11286和NJST258_1缺失;荚膜多糖基因簇中基因如糖基转移酶基因wzy、聚集蛋白基因wzi、荚膜转运蛋白基因wza等突变株在血清处理后不能检出,气杆菌素、沙门氏菌素等铁载体基因簇在各文库中的丰度变化不超过1倍;KEGG注释结果显示,注释最多的基因为参与氨基酸代谢、辅助因子和维生素代谢、碳水化合物代谢等。结论Tn-seq是功能基因筛选的可靠方法,本研究成功筛选出405个hvKp的候选新毒力基因,为后续深入研究hvKp的新毒力基因功能和调控机制提供实验依据。Objective To construct a transposon mutation library and screen new virulence genes of hypervirulent Klebsiella pneumoniae(hvKp).Methods The transposon mutation library was constructed and treated with human serum.The changes in the abundance of the genes of library mutant strains were analyzed by Transposon sequencing(Tn-seq).Besides,KEGG(kyoto encyclopedia of genes and genomes)annotation and enrichment analysis were performed on the screened genes.Results A total of 405 genes were screened out according to the abundance of the genes in the library treated with human serum was 20%lower than that without treated,and 351 genes,86.7%of these genes were conserved in HS11286,NJST258_1,NTUH-K2044 and RJF293.Ten genes existed in strains NTUH-K2044 and RJF293 with high virulence,while these genes were absent in HS11286 and NJST258_1 with low virulence.The mutants with genes such as glycosyl transferase gene wzy,aggregator protein gene wzi and capsule transporter gene wza,which belong to the capsule polysaccharide gene clusters,could not be detected after serum treatment.The abundance of iron carriers gene clusters such as aerobacterin and salmonellin in each library changed less than one time.KEGG annotation results showed that most annotated genes were involved in amino acid metabolism,cofactor and vitamin metabolism,carbohydrate metabolism,etc.Conclusions Tn-seq is a reliable method to screen functional genes.In this study,405 candidate virulence genes of hvKp were successfully screened out,providing an experimental basis for further research on the function and regulation mechanism of new virulence genes of hvKp.

关 键 词：转座子突变文库 血清杀菌实验 转座子测序 高毒力肺炎克雷伯菌 新毒力基因 

分 类 号：R378[医药卫生—病原生物学]