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 drug-resistance and virulence and the effect of IS600 on the expression of cse2 in Shigella

【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 m RNA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m RNA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 m RNA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 m RNA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 m RNA表达水平有影响。

[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 m RNA 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 m RNA 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 m RNA expression level of cse2 was decreased in Shigella.