title: Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.
abstract: 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.
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan; Center for Transdisciplinary Research, Niigata University, Niigata 950-2181, Japan. Electronic address: mtanig@eng.niigata-u.ac.jp.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Taniguchi
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Ochiai
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kondo
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Fukuda
authors.affiliation: Center for Fostering Innovative Leadership, Niigata University, Niigata 950-2181, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Ishiyama
authors.affiliation: Graduate School of Technology, Niigata Institute of Technology, Niigata 945-1195, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Saitoh
authors.affiliation: Department of Chemistry, Tokyo Dental College, Tokyo 101-0062, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Kato
authors.affiliation: Department of Materials Science and Technology, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000494:pharmacology*; D001761:Bleomycin / Q000494:pharmacology; D002474:Cell-Free System / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000737:chemistry* / Q000494:pharmacology*; D007313:Insecta / Q000737:chemistry*; D011392:Proline / Q000032:analysis*; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D015203:Reproducibility of Results; D013307:Streptomycin / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D029968:Escherichia coli Proteins; D019476:Insect Proteins; D012333:RNA, Messenger; D001761:Bleomycin; D049452:Green Fluorescent Proteins; C087710:pyrrhocoricin protein, Pyrrhocoris apterus; D011392:Proline; D013307:Streptomycin
keywords: Antimicrobial peptide; Cell-free translation system; Inhibition of protein synthesis; Intracellular target; Mechanism of action; Pyrrhocoricin