title: Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system.
abstract: Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.
authors.affiliation: Department of Biological Science and Technology, Science University of Tokyo, Yamazaki, Noda, Chiba 278-8510, Japan. staguchi@postman. riken.go.jp
authors.forename: S
authors.initials: S
authors.lastname: Taguchi
authors.forename: K
authors.initials: K
authors.lastname: Kuwasako
authors.forename: A
authors.initials: A
authors.lastname: Suenaga
authors.forename: M
authors.initials: M
authors.lastname: Okada
authors.forename: H
authors.initials: H
authors.lastname: Momose
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides; D001483:Base Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000187:drug effects*; D009682:Magnetic Resonance Spectroscopy; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010449:Peptide Mapping; D010456:Peptides, Cyclic / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D010456:Peptides, Cyclic; C098076:thanatin