Imaging the antimicrobial mechanism(s) of cathelicidin-2.
Host defence peptides (HDPs) have the potential to become alternatives to conventional antibiotics in human and veterinary medicine. The HDP chicken cathelicidin-2 (CATH-2) has immunomodulatory and direct killing activities at micromolar concentrations. In this study the mechanism of action of CATH-2 against Escherichia coli (E. coli) was investigated in great detail using a unique combination of imaging and biophysical techniques. Live-imaging with confocal fluorescence microscopy demonstrated that FITC-labelled CATH-2 mainly localized at the membrane of E. coli. Upon binding, the bacterial membrane was readily permeabilized as was shown by propidium iodide influx into the cell. Concentration- and time-dependent effects of the peptide on E. coli cells were examined by transmission electron microscopy (TEM). CATH-2 treatment was found to induce dose-dependent morphological changes in E. coli. At sub-minimal inhibitory concentrations (sub-MIC), intracellular granulation, enhanced vesicle release and wrinkled membranes were observed, while membrane breakage and cell lysis occurred at MIC values. These effects were visible within 1-5 minute of peptide exposure. Immuno-gold TEM showed CATH-2 binding to bacterial membranes. At sub-MIC values the peptide rapidly localized intracellularly without visible membrane permeabilization. It is concluded that CATH-2 has detrimental effects on E. coli at concentrations that do not immediately kill the bacteria.
Department of Infectious Diseases and Immunology, Division Molecular Host Defence, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Viktoria A F
VA
Schneider
Department of Infectious Diseases and Immunology, Division Molecular Host Defence, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Maarten
M
Coorens
Department of Infectious Diseases and Immunology, Division Molecular Host Defence, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Soledad R
SR
Ordonez
Department of Infectious Diseases and Immunology, Division Molecular Host Defence, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Johanna L M
JL
Tjeerdsma-van Bokhoven
Department of Cell Biology, Cell Microscopy Core, University Medical Centre Utrecht, Utrecht, The Netherlands.
George
G
Posthuma
Department of Infectious Diseases and Immunology, Division Molecular Host Defence, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Albert
A
van Dijk
Department of Infectious Diseases and Immunology, Division Molecular Host Defence, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Henk P
HP
Haagsman
Department of Infectious Diseases and Immunology, Division Molecular Host Defence, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Edwin J A
EJ
Veldhuizen
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology*; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects* / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D059010:Single-Cell Analysis / Q000379:methods*
D023181:Antimicrobial Cationic Peptides; C504855:CMAP27 protein, chicken; D016650:Fluorescein-5-isothiocyanate