Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system

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• 作者：Masayuki Taniguchi¹ ; ² ; ^{mtanig@eng.niigata-u.ac.jp" class="auth_mail" title="E-mail the corresponding author} ; Akihito Ochiai¹ ; Hiroshi Kondo¹ ; Shun Fukuda¹ ; Yohei Ishiyama³ ; Eiichi Saitoh⁴ ; Tetsuo Kato⁵ ; Takaaki Tanaka¹
• 关键词：Pyrrhocoricin ; Antimicrobial peptide ; Cell-free translation system ; Mechanism of action ; Intracellular target ; Inhibition of protein synthesis
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：2016
• 出版时间：May 2016
• 年：2016
• 卷：121
• 期：5
• 页码：591-598
• 全文大小：668 K

文摘

Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step.