sRNA (istR)在肠炎沙门氏菌抗活性氮氧中的作用分析

Analysis of regulation role of sRNA (istR) in Salmonella resistant to reactive nitrogen and oxygen intermediates

摘要:

sRNA (Small non-coding RNA, sRNA)为新近发现的基因表达调控分子, 转录后水平调控靶基因表达, 在细菌毒力、应激及对外界环境感应方面起调控作用。沙门氏菌是一种重要的人畜共患病病原菌, 可引起人类食物中毒、败血症及伤寒等。以肠炎型沙门氏菌为模型, 研究sRNA (istR)在肠炎沙门氏菌抗活性氮氧中的作用, 为沙门氏菌的防治提供新方向。参考已报道的沙门氏菌全基因组及istR序列, 设计引物, PCR扩增istR突变用基因片段, 运用Red同源重组系统对肠炎沙门氏菌(SE2472)的istR基因进行定点敲除, 构建敲除菌株(SE2472△istR), 比较野生株和敲除株对活性氮氧的敏感性; 构建回复表达质粒pHDB3-istR, 将其转入istR敲除株构建回复株SE2472△istR-comp, 以回复表达istR, 分析istR表达对沙门氏菌istR敲除株抵抗活性氮氧的回复作用。活性氮抑菌结果表明, SE2472在pH 5.0、NaNO2浓度为20 mmol/L的LB液体培养基中培养3 h, 存活率为20.40%; 培养6 h, 存活率降至0.05%。同等培养条件下, SE2472△istR的存活率分别为0.70%和0, SE2472△istR-comp生长情况与SE2472类似, 存活率分别为21.40%和0.08%。同时用H2O2分析istR在沙门氏菌抗活性氧中的作用, 活性氧抑菌结果表明, SE2472和SE2472△istR两者对H2O2的抑菌作用无明显差异。综合上述结果, 推测istR在沙门氏菌抗活性氮中起着调控作用, 在抗活性氧作用中没有调控作用。

Abstract:

Salmonella is a facultative intracellular pathogen that is associated with gastroenteritis, septicemia, and typhoid fever. It survives and replicates in macrophages during the course of infection and can be exposed to a number of stressful environments during its life cycle. Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI) which belong to oxygen-dependent antimicrobial systems of phagocytic cells. Resistance to ROI and RNI is a key feature of Salmonella virulence in vivo. Bacterial small non-coding RNA (sRNA) plays diverse physiological roles in stress responses, regulation of metabolism, control of bacterial envelope composition and bacterial virulence. In this study, we studied regulatory function of salmonella sRNA istR associated with resistance of S. enterica serovar Enteritidis to RNI and ROI. We constructed an istR-deletion mutant of SE2472 (SE2472△istR) using the red recombination system. The △istR mutant of SE2472 was complemented by cloned the wild-type allele of istR into plasmid pHDB3. To study the contribution of istR to the resistance of Salmonella enteritidis to RNI and ROI, we studied the differences of the survival rate of wild type SE2472, SE2472△istR mutant and complement strain to NaNO2 (pH 5.0, 20 mmol/L) and H2O2. The SE2472△istR was more sensitive to the bactericidal activity of NaNO2 (pH 5.0, 20 mmol/L) than wild type SE2472, but there is no difference in survival rate in the present of H2O2. To confirm the results, we constructed the complement plasmid and transferred it into SE2472△istR to rescue the survival defect. Results showed that the survival defect of SE2472△istR was rescued well when complement strain grown with NaNO2. The study of survival rate of the wild type SE2472 and istR deletion mutant in the presence of NaNO2 (pH 5.0, 20 mmol/L) and H2O2 demonstrated that sRNA istR acts as an important regulator of the resistance of SE oxygen-dependent antimicrobial systems.