反义锁核酸阻断耐甲氧西林金黄色葡萄球菌agr群体感应系统

Blocking Methicilli-Resistant Staphylococcus Aureus Agr Quorom Sensing System by Antisense LNA

目的:利用反义策略,设计、筛选针对agrA mRNA的反义锁核酸,阻断agr群体感应系统,降低MRSA毒力因子的表达,减小细菌生存压力,为抗耐药菌感染提供一种新策略。方法:应用Primer Premier 5.0和RNA structure 4.5两种软件,设计、筛选2条针对agrA mRNA的反义寡核苷酸序列,对其进行锁核酸修饰,并与透膜肽(KFF)3K共价连接,将这两条反义序列导入细菌体内。体外与MRSA共培养,观察细菌生存情况。实时定量PCR检测其对agrA及agr群体感受系统的效应分子RNAⅢ和下游毒力基因hla的转录水平的影响,Western blot检测其是否能抑制α-溶血素的表达。结果:两条反义锁核酸序列PLNA34和PLNA522在体外均无抗菌活性,但都能不同程度的抑制agrA,RNAⅢ和hla的转录水平;相比较而言,PLNA34的抑制效果更佳,并能明显抑制α-溶血素的分泌表达。结论:agrA可作为抗MRSA感染的新靶点,为新型抗MRSA药物的研发提供了理论基础。

Objective： Using antisense strategy to design and synthesize oligonucleotide targeting agrA mRNA to block agr quorum sensing system, so as to depress the virulence gene of MRSA. Methods： We design and select effective antisense locked nucleic acid （LNA） targeting agrA mRNA by software Primer Premier 5.0 and RNA structure 4. 5 and covalently conjugated LNA with the cell penetrating peptide （ KFF ）3K. The transcription of agrA and downstream virulence genes were analyzed by real-time quantitative PCR and the expression of α-toxin was detected by western blot. Results ： Neither PLNA34 nor PLNA522 inhibit the growth of MRSA in vitro. Both of PLNA34 and PLNA522 can significantly reduce the transcription of agrA, the effector RNAⅢ and hla, with PLNA34 having more significant inhibitory effect. PLNA34 has a stronger inhibition in expression of α-toxin than PLNA522. Conclusion： agrA could be a promising drug target to combat MRSA infection.