Entry of a Six-Residue Antimicrobial Peptide Derived from Lactoferricin B into Single Vesicles and Escherichia coli Cells without Damaging their Membranes

• Published in: Biochemistry (Easton) Vol. 56; no. 33; pp. 4419 - 4431
• Main Authors: Moniruzzaman, Md, Islam, Md. Zahidul, Sharmin, Sabrina, Dohra, Hideo, Yamazaki, Masahito
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.08.2017
• Subjects: Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - pharmacology; Cell Membrane - metabolism; Cytoplasm - metabolism; Escherichia coli - metabolism; Lactoferrin - chemistry; Lactoferrin - pharmacology; Liposomes; Phosphatidylcholines - chemistry; Phosphatidylcholines - pharmacology; Phosphatidylglycerols - chemistry; Phosphatidylglycerols - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.6b01274

Lactoferricin B (LfcinB) and shorter versions of this peptide have antimicrobial activity. However, the elementary processes of interactions of these peptides with lipid membranes and bacteria are still not well understood. To elucidate the mechanism of their antimicrobial activity, we investigated the interactions of LfcinB (4–9) (its sequence of RRWQWR) with Escherichia coli cells and giant unilamellar vesicles (GUVs). LfcinB (4–9) and lissamine rhodamine B red-labeled LfcinB (4–9) (Rh-LfcinB (4–9)) did not induce an influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli cells into their cytoplasm, indicating that no damage occurred in their plasma membrane. To examine the activity of LfcinB (4–9) to enter E. coli cytoplasm, we investigated the interaction of Rh-LfcinB (4–9) with single cells of E. coli containing calcein using confocal microscopy. We found that Rh-LfcinB (4–9) entered the cytoplasm without leakage of calcein. Next, we investigated the interactions of Rh-LfcinB (4–9) with single GUVs of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) mixtures containing a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using the single GUV method. The results indicate that Rh-LfcinB (4–9) outside the GUV translocated through the GUV membrane and entered its lumen without leakage of AF647. Interaction of Rh-LfcinB (4–9) with DNA increased its fluorescence intensity greatly. Therefore, we can conclude that Rh-LfcinB (4–9) can translocate across lipid membrane regions of the plasma membrane of E. coli cells to enter their cytoplasm without leakage of calcein and its antimicrobial activity is not due to damage of their plasma membranes.