The antimicrobial peptide Polybia-MP1 differentiates membranes with the hopanoid, diplopterol from those with cholesterol

Abstract

Polybia-MP1 is an antimicrobial peptide that shows a decreased activity in membranes with cholesterol (CHO). Since it is now accepted that hopanoids act as sterol-surrogates in some sterol-lacking bacteria, we here inquire about the impact of Polybia-MP1 on membranes containing the hopanoid diplopterol (DP) in comparison to membranes with CHO. We found that, despite the properties induced on lipid membranes by DP are similar to those induced by CHO, the effect of Polybia-MP1 on membranes with CHO or DP was significantly different. DP did not prevent dye release from LUVs, nor the insertion of Polybia-MP1 into monolayers, and peptide-membrane affinity was higher for those with DP than with CHO. Zeta potentials ( $\zeta$ ) for DP-containing LUVs showed a complex behavior at increasing peptide concentration. The effect of the peptide on membrane elasticity, investigated by nanotube retraction experiments, showed that peptide addition softened all membrane compositions, but membranes with DP got stiffer at long times. Considering this, and the $\zeta$ results, we propose that peptides accumulate at the interface adopting different arrangements, leading to a non-monotonic behavior. Possible correlations with cell membranes were inquired testing the antimicrobial activity of Polybia-MP1 against hopanoid-lacking bacteria pre-incubated with DP or CHO. The fraction of surviving cells was lower in cultures incubated with DP compared to those incubated with CHO. We propose that the higher activity of Polybia-MP1 against some bacteria compared to mammalian cells is not only related to membrane electrostatics, but also the composition of neutral lipids, particularly the hopanoids, could be important.

Keywords: Bacterial resistance; Lytic activity; Membrane dynamic elasticity; Optical tweezers; Targeting of AMPs; Zeta potential.

Graphical abstract

Fig. 1

(A) Area change supported by lipid monolayers due to peptide insertion. Film composition: pure POPC (black), POPC + 20$\%$ (blue) or 40$\%$ (cyan) of CHO, or POPC + 20$\%$ (olive) or 40$\%$ (green) of DP. All data correspond to the average ($\pm$ SD) of at least three independent experiments. * indicates significant statistical differences determined by one-way ANOVA, Tukey’s test at $p<0.05$. (B) Lytic activity of the peptide. Fluorescence of released CF from LUVs composed of pure POPC (black), POPC + 20% CHO (blue), or POPC + 20% DP (olive). The vertical lines represent the [P]/[L]${}_{50}$ values. All data correspond to the average ($\pm$ SD) of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

(A)Kinetic curves for the fluorescence intensity signal of MP1 labeled with FITC (fMP1) at the rim (magenta), at the lumen (orange), and outside a vesicle (violet) normalized by the final fluorescence outside the vesicle. GUV composition: POPC + 0.5 mol% of PE-Rho in the presence of 0.6 µM fMP1. (B) representative confocal images at selected times after peptide addition. Upper sequence: red channel (PE-Rho), lower sequence: green channel (fMP1). Scale bar: 10 µm. For better visualization of the images, the brightness and contrast range was reduced from an original range of 0--255 to 0--132. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

(A) Final fluorescence intensity of fMP1 at the membrane rim normalized by the intensity outside the GUVs ($(F_M$/$F_0){}_{\max }$) (filled bars, left scale); and ratio between $k_a$ and $k_d$ ($K_{ef}$), see text for details (open bars, right scale). Data correspond to the average ($\pm$ SD) of at least 30 GUVs for $(F_M$/$F_0){}_{\max }$) or 15 for $K_{ef}$. (B) Times for achieving half the value of $(F_M$/$F_0){}_{\max }$ at the vesicle’s rim (filled bars), and times at which peptide fluorescence is detected at the lumen of 50% of the vesicles (open bars). All data correspond to the average ($\pm$ SD) of at least 45 GUVs. Different letters indicate significant differences analyzed by one-way ANOVA and Tukey’s test at $p<0.05$.

Fig. 4

Confocal images at selected times after addition of 6.0 µM fMP1 to POPC vesicles (upper panels) or POPC + 20% DP vesicles (lower panels). The images correspond to the merge of green (fMP1) and red (PE-Rho) channels. Brightness and contrast range: 0–150, scale bar: 10 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Membrane dynamic elasticity. (A) Retraction process of tethers pulled from a POPC GUV after 0, 10, 13, 17, and 25 min of peptide addition. The solid lines represent fits to the data using $L=L_0\exp (\frac{-t}{\tau }$), where $\tau$ is the characteristic time. Inset: accumulated images obtained using DIC showing the bead motion (24 frames/s) after the laser was switched off. Left: without peptide, right: after 17 min of MP1 addition, note the slower bead motion and the loss of vesicle contrast due to the increased permeability. (B) Characteristic times $\tau$ as a function of time after the addition of 0.6 µM peptide to different GUVs composed of POPC + 20% CHO (each symbol correspond to a single GUV) (C) Characteristic times $\tau$ as a function of time after the addition of 0.6 µM peptide to GUVs composed of POPC (black), POPC + 20% DP (olive), POPC + 40% CHO (cyan) or DP (green). Data correspond to the average ($\pm$ SD) of at least 5 GUVs from at least two independent experiments. * and ** indicate significant statistical differences between the values at the indicated times and at the start and end of the experiment determined by one-way ANOVA, Tukey’s test at $p<0.02$ or $p<0.002,$ respectively.

Fig. 6

Effect of MP1 on the zeta potential ($\zeta$) of vesicles composed of POPC + 20$\%$ (olive) or 40$\%$ of DP (green). All data correspond to the average ($\pm$ SD) of three independent experiments. Inset: wheel representation of MP1 in an $\alpha$-helix configuration (green represents positively charged; red, negatively charged; blue, polar uncharged and yellow, hydrophobic residues, and N and C indicate the N-terminus and C-terminus of the peptide, respectively.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Activity of MP1 against P. aeruginosa previously exposed to CHO or DP. Fraction of surviving cells without treatment (control 1) or incubated in the solvent (control 2), or in a solution of CHO (blue) or DP (olive) after 3 h of exposure to 9 µM or 18 µM of MP1. All data correspond to the average ($\pm$ SD) of three independent experiments. * and ** indicate significant statistical differences determined by one-way ANOVA, Tukey’s test at $p<0.0$5 or $p<0.001,$ respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)