title: Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli.
abstract: The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time-dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 microM. Bactericidal kinetics at the MIC indicated rapid killing of E.coli (<30 min). Membrane permeability studies illustrated permeation as a time-dependent event. Maximum permeability at the MIC occurred within 30 min, which correlates to the bactericidal action. Further investigation showed that the immediate permeabilizing action of CP1 is concentration-dependent, which correlates to the concentration-dependent nature of the inhibition assays. At the MIC and above, the immediate permeability was significant enough that the cells could not recover and exhibit growth. Below the MIC, immediate permeability was evident, but the level was insufficient to inhibit growth. Dansyl polymyxin B displacement studies showed LPS binding is essentially the same at all concentrations investigated. However, it does appear that only the immediate interaction is important, because binding continued to increase over time beyond cell viability. Our studies correlated CP1 bactericidal kinetics to membrane permeability suggesting CP1 concentration-dependent killing is driven by the extent of the immediate permeabilizing action of the peptide.
authors.affiliation: Biological Science and Technology Team, Warfighter Science Technology and Applied Research Directorate, U.S. Army Natick Soldier Research, Development, & Engineering Center, Natick, MA 01760-5020, USA. steven.arcidiacono@us.army.mil
authors.forename: Steven
authors.initials: S
authors.lastname: Arcidiacono
authors.forename: Jason W
authors.initials: JW
authors.lastname: Soares
authors.forename: Alexa M
authors.initials: AM
authors.lastname: Meehan
authors.forename: Patrick
authors.initials: P
authors.lastname: Marek
authors.forename: Romy
authors.initials: R
authors.lastname: Kirby
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003124:Colorimetry; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry*; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011112:Polymyxin B / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000890:Anti-Infective Agents; D008070:Lipopolysaccharides; D010455:Peptides; D011112:Polymyxin B; C070016:cecropin P1-LI