CRISPR/Cas系统与志贺菌毒力和耐药的关系及插入序列IS600对cse2表达水平的影响

Role of CRISPR/Cas systems in drugresistance and virulence and the effect of IS600 on the expression of cse2 in Shigella

摘要:

[目的] 了解志贺菌中成簇的规律间隔短回文重复序列（Clustered regularly interspaced short palindromic repeats，CRISPR）的分布及其与毒力和耐药的关系，并分析志贺菌中插入序列IS600对CRISPR相关蛋白基因cse2 mRNA表达水平的影响。[方法] 利用课题组前期设计的引物PCR扩增志贺菌的3个CRISPR位点、CRISPR相关蛋白基因cse2、耐药基因和毒力基因；改良Kirby-Bauer （K-B）纸片法进行药敏试验；台盼蓝计数试验检测细菌毒力；Real-time PCR检测志贺菌中cse2基因mRNA表达水平。分别分析志贺菌中CRISPR/Cas系统与耐药基因、耐药表型、毒力基因、毒力表型的关系；了解IS600对CRISPR相关蛋白基因cse2 mRNA表达水平的影响。[结果] 志贺菌中CRISPR1位点阴性细菌的毒力强；插入序列IS600使cse2 mRNA表达水平降低。[结论] 志贺菌中存在CRISPR1、2、3位点；CRISPR1位点与毒力有关；插入序列IS600对cse2 mRNA表达水平有影响。

关键词:志贺菌;CRISPR/Cas系统;插入序列;耐药;毒力

Abstract:

[Objective] To analyze the relationship between CRISPR/Cas system and drug-resistance, virulence. To investigate the effect of IS600 on the expression of CRISPR associated gene cse2 in Shigella.[Methods] CRISPR loci, CRISPR associated gene cse2, drug-resistant genes and virulent genes were detected by PCR in 33 Shigella strains; Trypan Blue counting test was used to detect bacterial virulence; Real-time PCR was used to detect relative mRNA expression of cse2; susceptibilities of Shigella strains were tested by agar diffusion method. Furthermore, we analyzed the relationship between CRISPR loci and drug-resistant genes, virulent genes. The effect of the IS600 on the expression of CRISPR associated gene cse2 was investigated.[Results] The mortality of Hela cells infected by Shigella with CRISPR1 loci was significantly lower (P<0.05) than those infected by Shigella without CRISPR1. The mRNA expression level of cse2 in group with IS600 was significantly (P<0.05) lower than that in group without IS600.[Conclusions] CRISPR loci were widely present in Shigella. Shigella without CRISPR1 has a higher pathogenicity. Due to the insertion of IS600, the mRNA expression level of cse2 was decreased in Shigella.

Key words:Shigella;CRISPR/Cas system;insertion sequence;drug-resistance;virulence