1-2-oleoylphosphatidylcholine and Escherichia-coli-Infections

Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to the surface charges. Analysis of Lfcin B-induced shape changes indicated that the binding of Lfcin B increased the area of the outer monolayer of GUVs. These results indicate that Lfcin B-induced damage of the plasma membrane of E. coli with its concomitant rapid leakage of internal contents is a key factor for the bactericidal activity of LfcinB.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Cattle; Cell Membrane Permeability; Escherichia coli; Escherichia coli Infections; Fluoresceins; Fluorescent Dyes; Humans; Lactoferrin; Molecular Sequence Data; Organic Chemicals; Phosphatidylcholines; Phosphatidylglycerols; Static Electricity; Sucrose; Unilamellar Liposomes

Amphiphilic methacrylate co-polymers recently demonstrated antimicrobial activity. To understand their activity mechanism, we prepared three homologous methacrylate co-polymers with activity ranging from inactive (MMA) over specifically active (EMA) to non-specifically active (BMA) against bacteria and human erythrocytes. Fluorescent dye leakage assays were used to characterize their membrane-disrupting activity against liposomes of different compositions. From bacterial membrane-mimicking liposomes (composed of Escherichia coli extract or 20:80 DOPG/DOPE), the two active forms, EMA and BMA, caused more dye leakage than the inactive MMA, which mirrors their antibacterial activity trend. From mammalian membrane-mimicking liposomes (composed of DOPC or 20:80 DOPG/DOPC), the highly hemolytic BMA caused significantly more leakage than MMA and EMA, which mirrors its hemolytic activity trend. Moreover, to dissect the effect of intrinsic membrane curvature from that of membrane charge, we used a ternary membrane with constant charge and tunable intrinsic curvature. Specifically, we used membranes composed of DOPG/DOPE/DOPC with constant DOPG content and varying DOPE/DOPC ratio. To significantly disrupt this model, methacrylate co-polymers with different activity profiles required a different minimum threshold DOPE content. In contrast, variation in DOPG/DOPC ratio at constant DOPE concentration did not show a similar influence on the selective membrane-disrupting activity of these co-polymers. Our results suggested that the intrinsic membrane curvature, rather than membrane charge, may play a major role in the selective membrane-disrupting activity of methacrylate co-polymers. Since more PE lipids exist in bacterial membranes than in eukaryotic membranes, our results imply that negative-intrinsic-curvature lipids such as PE may contribute to the selective antimicrobial activity.

Topics: Animals; Anti-Bacterial Agents; Escherichia coli; Escherichia coli Infections; Humans; Liposomes; Methacrylates; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polymers