Effects of short interfering RNA against methicillin-resistant Staphylococcus aureus coagulase in vitro and in vivo
Effects of short interfering RNA against methicillin-resistant Staphylococcus aureus coagulase in vitro and in vivo
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DOI：
10.1093/jac/dki416
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发表时间：
2006-01-01
影响因子：
5.2
通讯作者：
Kohno, S
中科院分区：
文献类型：
--
作者：
Yanagihara, K;Tashiro, M;Kohno, S
目标：金黄色葡萄球菌等抗生素耐药性细菌的出现需要创造性的研究和开发策略。在这方面，抑制细菌发病机制可能是一种有前途的治疗方法。短干扰RNA（siRNA）的基因沉默效应对于该策略是有用的。我们研究了siRNA对凝固酶表达的影响，因为凝固酶是耐甲氧西林链球菌发病机制中最重要的酶之一。方法：设计并合成针对金黄色葡萄球菌凝固酶的21 bp siRNA双链体。进行RT-PCR以确定siRNA是否抑制凝固酶mRNA的表达，并使用放射性标记的siRNA来确认体外转染细菌。结果：RT-PCR结果显示，siRNA能显著抑制凝血酶mRNA的表达。siRNA组和对照组的凝固酶滴度分别为8和32。掺入的放射性的测量表明siRNA被递送到细菌中。在鼠感染模型中，对照组和siRNA组分别为7.64 +/- 0.42和6.29 +/- 0.23 log cfu/mL，（平均值+/- SEM）分别检测到耐甲氧西林金黄色葡萄球菌，表明siRNA组中活菌数量显着减少结论：siRNA在体外可抑制MRSA凝固酶mRNA的表达和凝固酶活性。体内结果显示，siRNA在降低血源性肺部感染的鼠模型中的细菌负荷方面是有效的。用siRNA靶向凝固酶似乎是治疗MRSA感染的新策略。
Objectives: The emergence of antibiotic-resistant bacteria such as Staphylococcus aureus calls for inventive research and development strategies. Inhibition of bacterial pathogenesis may be a promising therapeutic approach in this regard. The gene-silencing effect of short interfering RNA (siRNA) is useful for this strategy. We investigated the efficacy of siRNA on the expression of coagulase because it is the one of the most important enzymes in the pathogenesis of methicillin-resistant S. aureus (MRSA) infection.Methods: We designed and synthesized 21 bp siRNA duplexes against staphylococcal coagulase. RT-PCR was performed to determine whether the siRNAs inhibit the expression of the coagulase mRNA and radio-labelled siRNA was used to confirm transfection to bacteria in vitro. The efficacy of siRNA was determined in a murine model of haematogenous pulmonary infection.Results: RT-PCR showed that siRNAs significantly inhibited the expression of the coagulase mRNA. The coagulase titres in the siRNA and control groups were 8 and 32, respectively. Measurement of incorporated radioactivity indicated that the siRNAs were delivered into the bacteria. In the murine infection model, in control and siRNA groups, 7.64 +/- 0.42 and 6.29 +/- 0.23 log cfu/mL (mean +/- SEM) MRSA were detected, respectively, showing that there was a significant decrease in the number of viable bacteria in the siRNA group (P < 0.05).Conclusions: The results show that siRNA inhibited both mRNA expression and the activity of MRSA coagulase in vitro. The in vivo results revealed that the siRNA was effective in reducing the bacterial load in a murine model of haematogenous pulmonary infection. Targeting of coagulase with siRNA appears to be a novel strategy for treating MRSA infections.