title: Cationic amphipathic peptides KT2 and RT2 are taken up into bacterial cells and kill planktonic and biofilm bacteria.
abstract: We investigated the mechanisms of two tryptophan-rich antibacterial peptides (KT2 and RT2) obtained in a previous optimization screen for increased killing of both Gram-negative and Gram-positive bacteria pathogens. At their minimal inhibitory concentrations (MICs), these peptides completely killed cells of multidrug-resistant, enterohemorrhagic pathogen Escherichia coli O157:H7 within 1-5 min. In addition, both peptides exhibited anti-biofilm activity at sub-MIC levels. Indeed, these peptides prevented biofilm formation and triggered killing of cells in mature E. coli O157:H7 biofilms at 1 μM. Both peptides bound to bacterial surface LPS as assessed using the dansyl-polymyxin displacement assay, and were able to interact with the lipids of liposomes as determined by observing a tryptophan blue shift. Interestingly, even though these peptides were highly antimicrobial, they did not induce pore formation or aggregates in bacterial cell membranes. Instead these peptides readily penetrated into bacterial cells as determined by confocal microscopy of labeled peptides. DNA binding assays indicated that both peptides bound to DNA with higher affinity than the positive control peptide buforin II. We propose that cationic peptides KT2 and RT2 bind to negatively-charged LPS to enable self-promoted uptake and, subsequently interact with cytoplasmic membrane phospholipids through their hydrophobic domains enabling translocation across the bacterial membrane and entry into cells within minutes and binding to DNA and other cytoplasmic membrane. Due to their dual antimicrobial and anti-biofilm activities, these peptides may find use as an alternative to (or in conjunction with) conventional antibiotics to treat acute infections caused by planktonic bacteria and chronic, biofilm-related infections.
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand; Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand.
authors.forename: Thitiporn
authors.initials: T
authors.lastname: Anunthawan
authors.affiliation: Department of Microbiology and Immunology, Centre for Microbial Diseases and Immunity Research, 232-2259 Lower Mall Research Station, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
authors.forename: César
authors.initials: C
authors.lastname: de la Fuente-Núñez
authors.affiliation: Department of Microbiology and Immunology, Centre for Microbial Diseases and Immunity Research, 232-2259 Lower Mall Research Station, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. Electronic address: bob@hancocklab.com.
authors.forename: Robert E W
authors.initials: RE
authors.lastname: Hancock
authors.affiliation: Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand; Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Khon Kaen University, Khon Kaen, 40002, Thailand. Electronic address: somkly@kku.ac.th.
authors.forename: Sompong
authors.initials: S
authors.lastname: Klaynongsruang
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects*; D002412:Cations; D002462:Cell Membrane / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects*; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D008563:Membrane Lipids / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D010933:Plankton / Q000187:drug effects*; D014364:Tryptophan / Q000378:metabolism
chemical_list: D023181:Antimicrobial Cationic Peptides; D002412:Cations; D008070:Lipopolysaccharides; D008081:Liposomes; D008563:Membrane Lipids; C000600689:cationic amphipathic peptide KT2; C000600690:cationic amphipathic peptide RT2; D014364:Tryptophan; D004247:DNA
keywords: Antibacterial mechanism; Antibacterial peptide; Antibiofilm peptide; DNA binding; KT2; RT2