Structure and association of human lactoferrin peptides with Escherichia coli lipopolysaccharide

Abstract

An 11-amino-acid amphipathic synthetic peptide homologous to a helical region on helix 1 of human lactoferrin HLP-2 exhibited bactericidal activity against Escherichia coli serotype O111, whereas an analogue synthesized with Pro substituted for Met, HLP-6, had greatly reduced antimicrobial activity. The bactericidal activity of HLP-2 was 10-fold greater than that of HLP-6 in both buffer and growth medium by time-kill assays. These assays also showed a pronounced lag phase that was both concentration and time dependent and that was far greater for HLP-2 than for HLP-6. Both peptides, however, were shown to be equally efficient in destabilizing the outer membrane when the hydrophobic probe 1-N-phenylnaphthylamine was used and to have the same lipopolysaccharide (LPS) binding affinity, as shown by polymyxin B displacement. Circular dichroism (CD) spectroscopy was used to study the structure and the organization of the peptides in solution and upon interaction with E. coli LPS. In the presence of LPS, HLP-2 and HLP-6 were found to bind and adopt a beta-strand conformation rather than an alpha-helix, as shown by nonimmobilized ligand interaction assay-CD spectroscopy. Furthermore, this assay was used to show that there is a time-dependent association of peptide that results in an ordered formation of peptide aggregates. The rate of interpeptide association was far greater in HLP-2 LPS than in HLP-6 LPS, which was consistent with the lag phase observed on the killing curves. These results allow us to propose a mechanism by which HLP-2 folds and self-assembles at the outer membrane surface before exerting its activity.

FIG. 1.

Antibacterial effects of various HLP-2 and HLP-6 concentrations on E. coli in suspension medium. The residual viable E. coli cells were monitored in the presence of various HLP-2 (♦) and HLP-6 (•) concentrations as the numbers of CFU per milliliter, and the results were compared to those for control cells at time zero. Peptides were added to washed cells in PBS, with cell counts corresponding to 10⁸ cells ml⁻¹. Cell viability was determined after 2 h of incubation at 37°C. Each datum is the mean of at least three experiments, and each experiment was carried out in triplicate. Error bars are plotted as standard errors of the means.

FIG. 2.

Antibacterial effects of HLP-2 and HLP-6 on E. coli at timed intervals in suspension medium and growth medium. The residual viable E. coli cells were monitored at time intervals as the numbers of CFU per milliliter and compared to those for control cells (▪). (A) Effects of sublethal concentrations of HLP-2 over time. Peptide was added to washed cells corresponding to 10⁸ cells ml⁻¹ in PBS to a final concentration of 129 (•), 258 (▴), or 323 (▾) μM; and the effects on viability were determined at timed intervals. (B) Effects of lethal and sublethal concentrations of HLP-6 over time. Peptide was added to washed cells in PBS to a final concentration of 330 (•), 660 (▴), or 990 (▾) μM; and the effects on viability were determined at timed intervals. (C) Effects of lethal and sublethal concentrations of HLP-2 and HLP-6 on actively growing cells. Peptides were added to cultures in culture medium (1% proteose peptone), with cell counts corresponding to 5 × 10⁵ cells ml⁻¹, to a final concentration of 125 (•) or 250 (▴) μM for HLP-2 or 660 (▾) or 990 (♦) μM for HLP-6; and the effects on viability were monitored over time. Each datum is the mean of at least three experiments, and each experiment was carried out in triplicate. Error bars are plotted as standard errors of the means.

FIG. 3.

(A) Permeabilization of the E. coli outer membrane by HLP-2 and HLP-6 in the presence of NPN. Peptides were sequentially added to cells, and changes in the fluorescence intensity of the NPN probe were monitored. •, HLP-2; ▾, HLP-6; ▪, control with peptide and NPN. (B) Displacement of DPX from E. coli LPS by HLP-2, HLP-6, and lactoferrin. Peptides were sequentially added to E. coli LPS in the presence of DPX, and changes in the fluorescence intensity of the probe were monitored. Results are expressed as the percentage of fluorescence compared to the fluorescence for LPS-DPX complexes alone. •, HLP-2; ▾, HLP-6; ▪, lactoferrin. Each datum is the mean ± standard deviation of three experiments, and in all cases the standard deviation was less than 10%. Error bars were therefore excluded for clarity.

FIG. 4.

CD spectra of HLP-2 (A) and HLP-6 (B) showing changes in structures in the presence of LPS. The spectra were obtained by subtracting the spectrum of LPS as a baseline from those for the LPS-HLP mixtures. The differences in the CD spectra are indicative of the β-strand-type conformations induced on the HLP peptides upon interaction with LPS. The peptide concentration was 130 μM, and the ratio of the LPS concentration to peptide concentration was 1:46.

FIG. 5.

Changes in CD spectra of HLP-2 (A) and HLP-6 (B) in the presence of LPS over time. The peptide concentration was 130 μM, and the ratio of the LPS concentration to peptide concentration was 1:468.

FIG. 6.

Effects of time and temperature on the formations of HLP-2 and HLP-6 in the presence of LPS, as shown by CD spectral changes at 235 nm.