JAC Advance Access published online on December 12, 2005
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dki416
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1 Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan; Department of Pharmacotherapeutics, Nagasaki University Graduate School of Pharmaceutical Sciences, Nagasaki, Japan
* To whom correspondence should be addressed. 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 radiolabelled 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.
Received June 16, 2005
Revised October 4, 2005
Accepted October 18, 2005
Original article
Effects of short interfering RNA against methicillin-resistant Staphylococcus aureus coagulase in vitro and in vivo
Katsunori Yanagihara 1 *,
Miwa Tashiro 2,
Yuichi Fukuda 3,
Hideaki Ohno 3,
Yasuhito Higashiyama 3,
Yoshitsugu Miyazaki 3,
Yoichi Hirakata 3,
Kazunori Tomono 3,
Yohei Mizuta 3,
Kazuhiro Tsukamoto 1,
and
Shigeru Kohno 4
2 Department of Pharmacotherapeutics, Nagasaki University Graduate School of Pharmaceutical Sciences, Nagasaki, Japan
3 Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan
4 Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan; Division of Molecular and Clinical Microbiology, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Medical Sciences, Nagasaki, Japan
Katsunori Yanagihara, E-mail: kyana-ngs{at}umin.ac.jp
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