title: Recombinant expression, antimicrobial activity and mechanism of action of tritrpticin analogs containing fluoro-tryptophan residues.
abstract: The increase in antibiotic-resistant bacterial infections has prompted significant academic research into new therapeutic agents targeted against these pathogens. Antimicrobial peptides (AMPs) appear as promising candidates, due their potent antimicrobial activity and their ubiquitous presence in almost all organisms. Tritrpticin is a member of this family of peptides and has been shown to exert a strong antimicrobial activity against several bacterial strains. Tritrpticin's main structural characteristic is the presence of three consecutive Trp residues at the center of the peptide. These residues play an important role in the activity of tritrpticin against Escherichia coli. In this work, a recombinant version of tritrpticin was produced in E. coli using calmodulin as a fusion protein expression tag to overcome the toxicity of the peptide. When used in combination with glyphosate, an inhibitor of the endogenous synthesis of aromatic amino acids, this expression system allowed for the incorporation of fluorinated Trp analogs at very high levels (>90%). The antimicrobial activity of the 4-, 5- and 6-fluoro-Trp-containing tritrpticins against E. coli was as strong as the activity of the native peptide. Similarly, the tritrpticin analogs exhibited comparable abilities to perturb and permeabilize synthetic lipid bilayers as well as the outer and inner membrane of E. coli. Furthermore, the use of 19F NMR spectroscopy established that each individual fluoro-Trp residue interacts differently with SDS micelles, supporting the idea that each Trp in the original tritrpticin plays a different role in the perturbing/permeabilizing activity of the peptide. Moreover, our work demonstrates that the use of fluoro-Trp in solvent perturbation 19F NMR experiments provides detailed site-specific information on the insertion of the Trp residues in biological membrane mimetics. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Arias
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Elesha R
authors.initials: ER
authors.lastname: Hoffarth
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, T2N 1N4, Canada.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ishida
authors.affiliation: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Aramini
authors.affiliation: Biochemistry Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, T2N 1N4, Canada. Electronic address: vogel@ucalgary.ca.
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Vogel
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism; D000097797:Glyphosate
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D002147:Calmodulin; D005998:Glycine; D008051:Lipid Bilayers; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D000097797:Glyphosate; C402895:tritrpticin
keywords: (19)F NMR; Antimicrobial peptides; Fluoro-tryptophan; Recombinant peptide; Tritrpticin