Abstract
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.