RNAⅢ抑制肽对不同生长时相金黄色葡萄球菌毒力因子表达的影响

Effect of RNAⅢ inhibitor peptide on the expression of virulence factors of Staphylococcus aureus at different growth phases

目的探究RNAⅢ抑制肽(RIP)在细菌的不同生长时相对葡萄球菌溶素和黏附相关蛋白表达水平的调节作用。方法按比例接种耐甲氧西林金黄色葡萄球菌至培养基中,将过夜培养的细菌分别转接至TSB培养基(含0.5%的葡萄糖),将其浓度调整至1×10^8 CFU/mL。RIP组加入课题组设计合成的RIP衍生物(RIP1183),对照组加入无菌磷酸盐缓冲液。检测其对细菌生长曲线和生物膜形成的影响,应用实时荧光定量聚合酶链式反应的方法,在细菌迟缓期(2 h)、对数期(6 h)和稳定期(10 h)检测RIP1183对RNAⅢ、人类白细胞抗原(HLA)、icaA和fnbA基因的转录水平的影响,并与对照组进行比较。结果与对照组比较,RIP1183不影响细菌生长,差异无统计学意义(P>0.05)。与对照组比较,RIP组细菌生物膜形成显著减少(P<0.01)。迟缓期(2 h),与对照组比较,RIP组RNAⅢ表达明显降低(P<0.05)。对数期(6 h),与对照组比较,RIP组RNAⅢ、HLA表达显著降低(P<0.01);稳定期(10 h),与对照组比较,RIP组icaA、fnbA和RNAⅢ表达显著降低(P<0.05或P<0.01)。结论RIP1183通过抑制附属基因调节系统的效应分子RNAⅢ的表达,显著降低不同时相不同毒力因子的表达,从而破坏细菌对环境适应机制,可能是其抗菌活性和抗生物膜形成的机制之一。

Objective To explore the role of RNAⅢ inhibitory peptide(RIP)in regulating the expression of staphylolysin and adhesion-related proteins at different growth phases.Methods The methicillin-resistant Staphylococcus aureus was inoculated to medium proportionally,and the bacteria cultured overnight were transferred to TSB medium(containing 0.5%glucose),respectively,and the concentration was adjusted to 1×10^8 CFU/mL.The RIP group was added with RIP derivatives(RIP1183)designed and synthesized by research group,while the control group was added with sterile phosphate buffer.Its effects on the growth curve and biofilm formation were examined.Quantitative real-time fluorescence polymerase chain reaction was used to detect the expression levels of RNAⅢ,human leucocyte antigen(HLA),icaA,and fnbA during the bacterial retardation phase(2 h),logarithmic phase(6 h),and stable phase(10 h),and compared with the control group.Results Compared with the control group,RIP1183 did not affect the growth of bacteria,and the difference was not statistically significant(P>0.05).Compared with the control group,the formation of bacterial biofilms in the RIP group was significantly reduced(P<0.01).In the bacterial retardation phase(2 h),the expression of RNAⅢ in RIP group was obviously decreased(P<0.05).In the logarithmic phase(6 h),compared with the control group,the expression of RNAⅢ and HLA were obviously decreased(P<0.01).In the stable phase(10 h),the expression of icaA,fnbA and RNAⅢwere obviously decreased(P<0.05 or P<0.01).Conclusion RIP1183 inhibits the expression of RNAⅢ,an effector molecule of the accessory gene regulation system,and significantly reduces the expression of different virulence factors in different phases,thereby destroying the mechanism of bacteria’s adaptation to the environment,which may be one of the mechanisms of its antibacterial activity and anti-biofilm formation.