Membrane potential is vital for rapid permeabilization of plasma membranes and lipid bilayers by the antimicrobial peptide lactoferricin B

Abstract

Edited by Karen G. Fleming Lactoferricin B (LfcinB) is a cationic antimicrobial peptide, and its capacity to damage the bacterial plasma membrane is suggested to be a main factor in LfcinB's antimicrobial activity. However, the specific processes and mechanisms in LfcinB-induced membrane damage are unclear. In this report, using confocal laser-scanning microscopy, we examined the interaction of LfcinB with single Escherichia coli cells and spheroplasts containing the water-soluble fluorescent probe calcein in the cytoplasm. LfcinB induced rapid calcein leakage from single E. coli cells and from single spheroplasts, indicating that LfcinB interacts directly with the plasma membrane and induces its rapid permeabilization. The proton ionophore carbonyl cyanide m-chlorophenylhydrazone suppressed this leakage. Next, we used the single giant unilamellar vesicle (GUV) method to examine LfcinB's interaction with GUVs comprising polar lipid extracts of E. coli containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). We observed that LfcinB stochastically induces local rupture in single GUVs, causing rapid AF647 leakage; however, higher LfcinB concentrations were required for AF647 leakage from GUVs than from E. coli cells and spheroplasts. To identify the reason for this difference, we examined the effect of membrane potential on LfcinB-induced pore formation, finding that the rate of LfcinB-induced local rupture in GUVs increases greatly with increasing negative membrane potential. These results indicate that membrane potential plays an important role in LfcinB-induced local rupture of lipid bilayers and rapid permeabilization of E. coli plasma membranes. On the basis of these results, we discuss the mode of action of LfcinB's antimicrobial activity.

Action of antimicrobial peptides and cell-penetrating peptides on membrane potential revealed by the single GUV method

“…local rupture of GUVs at φ m = − 102 mV. In contrast, the rate of membrane permeation of AF647 after local rupture at φ m = − 102 mV is almost the same as that at 0 mV (Hossain et al 2019). These results indicate that the φ m increases the rate constant of LfcinB-caused local rupture but does not change the rate constant of membrane permeation of AF647.…”

“…This rapid membrane permeation is different from the gradual membrane permeation of calcein via magainin 2-caused pores in lipid bilayers (Tamba and Yamazaki 2009). LfcinB also induces membrane permeation of calcein from single E. coli cells stochastically, for example, with 5-μM LfcinB, all examined single cells show membrane permeation of calcein with various rates within 6 min (Hossain et al 2019). However, 5-μM LfcinB cannot induce membrane permeation of fluorescent dye, Alexa Fluor 647 hydrazide (AF647), from most single GUVs comprising E. coli polar lipid extract (E. coli-lipid: phosphatidylethanolamine (PE)/phosphatidyl glycerol (PG)/cardiolipin (CA) [67/ 23/10 (wt% ratio)]).…”

“…In the current method for application of φ m to GUVs, the membrane contains a low concentration of gramicidin A (monovalent cation channel) (Finkelstein and Andersen 1981;Hille 1992), and K + concentration difference is produced between the inside and the outside of the GUVs (Fig. 1c) (Hossain et al 2019;Moghal et al 2020). To adjust the osmotic pressure, we use tetraethylammonium ion (TEA + ) so that the sum of K + and TEA + concentrations in aqueous solution outside the GUVs equals to K + concentration in the GUV lumen, because it is known that TEA + cannot pass through the gramicidin A channel without its blocking (Andersen 1983).…”

“…However, 5-μM LfcinB cannot induce membrane permeation of fluorescent dye, Alexa Fluor 647 hydrazide (AF647), from most single GUVs comprising E. coli polar lipid extract (E. coli-lipid: phosphatidylethanolamine (PE)/phosphatidyl glycerol (PG)/cardiolipin (CA) [67/ 23/10 (wt% ratio)]). To elucidate this discrepancy, the effect of φ m on the interaction of LfcinB with single E. coli-lipid-GUVs was studied using CLSM (Hossain et al 2019). Figure 2a shows the interaction of 5-μM LfcinB with a single E. coli-lipid-GUV containing AF647 at φ m = −102 mV.…”

Design, Engineering and Discovery of Novel α-Helical and β-Boomerang Antimicrobial Peptides against Drug Resistant Bacteria

“…Antibacterial peptides can be obtained from bacterial cell agglutinating eosinophil cationic protein (RNase 3) and potent analogs were further designed based on the structures [162,163]. An AMP termed lactoferricin derived from the N-terminal of iron binding protein lactoferrin and its shorter analogs were extensively characterized for antimicrobial activity and mechanism [164][165][166][167][168][169][170]. More recent studies have identified several fragments of ApoE protein with broad spectrum antimicrobial activity.…”