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. 2019 Oct 1;1861(10):182980.
doi: 10.1016/j.bbamem.2019.05.002. Epub 2019 May 5.

Hybrids made from antimicrobial peptides with different mechanisms of action show enhanced membrane permeabilization

Heidi M Wade  1 , Louise E O Darling  2 , Donald E Elmore  3
Affiliations

Hybrids made from antimicrobial peptides with different mechanisms of action show enhanced membrane permeabilization

Heidi M Wade et al. Biochim Biophys Acta Biomembr. .

Abstract

Combining two known antimicrobial peptides (AMPs) into a hybrid peptide is one promising avenue in the design of agents with increased antibacterial activity. However, very few previous studies have considered the effect of creating a hybrid from one AMP that permeabilizes membranes and another AMP that acts intracellularly after translocating across the membrane. Moreover, very few studies have systematically evaluated the order of parent peptides or the presence of linkers in the design of hybrid AMPs. Here, we use a combination of antibacterial measurements, cellular assays and semi-quantitative confocal microscopy to characterize the activity and mechanism for a library of sixteen hybrid peptides. These hybrids consist of permutations of two primarily membrane translocating peptides, buforin II and DesHDAP1, and two primarily membrane permeabilizing peptides, magainin 2 and parasin. For all hybrids, the permeabilizing peptide appeared to dominate the mechanism, with hybrids primarily killing bacteria through membrane permeabilization. We also observed increased hybrid activity when the permeabilizing parent peptide was placed at the N-terminus. Activity data also highlighted the potential value of considering AMP cocktails in addition to hybrid peptides. Together, these observations will guide future design efforts aiming to design more active hybrid AMPs.

Keywords: Antimicrobial peptide; Hybrid peptide; Membrane permeabilization; Membrane translocation; Peptide design.

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Figures

Figure 1:
Figure 1:
Antimicrobial activity of peptides against E. coli measured by radial diffusion assays. Hybrids were made from combining BF2 and magainin 2 (A), BF2 and parasin (B), DesHDAP1 and magainin 2 (C) or DesHDAP1 and parasin (D). Parent peptides are colored based on their primary mechanism of action; red for translocating and blue for membrane permeabilizing. Hybrid peptides are given the color of the more N-terminal peptide in the sequence. Data for an equimolar mixture of the two peptides (not in a covalently-linked hybrid) are denoted with a +. A minimum of 12 readings from at least 3 individual cultures were taken for each peptide. Symbols above bars indicate statistical significance (t-test, p<0.05) from the translocating parent (†), permeabilizing parent (*), equimolar mixture (‡), or the indicated peptide pair (#). Error bars represent the standard error of the mean.
Figure 2:
Figure 2:
Membrane permeabilization of peptides with E. coli measured by the propidium iodide (PI) assay. Permeabilization is shown as the average ratio of PI fluorescence 5 minutes after addition of peptides to cells (F5min) relative to initial fluorescence (Fo). Data is shown for hybrids made from combining BF2 and magainin 2 (A), BF2 and parasin (B), DesHDAP1 and magainin 2 (C) or DesHDAP1 and parasin (D). A minimum of 3 individual samples were measured for each average other than magainin+BF2 (n=2). Colors are as in Figure 1. Symbols above bars indicate statistical significance (t-test, p<0.05) from the translocating parent (†), permeabilizing parent (*), equimolar mixture (‡), or the indicated peptide pair (#). Error bars represent the standard error of the mean.
Figure 3:
Figure 3:
Cellular localization of peptides in E. coli spheroplasts measured by confocal microscopy. Data are shown as percentage of spheroplasts with internal peptide localization, or translocation into the spheroplasts. A minimum of 69 spheroplasts were visualized for each peptide. Data are shown for hybrids made from combining BF2 and magainin 2 (A), BF2 and parasin (B), DesHDAP1 and magainin 2 (C) or DesHDAP1 and parasin (D). Colors are as in Figure 1. Symbols above bars indicate statistical significance (chi-square test, p<0.05) from the translocating parent (†) or permeabilizing parent (*).
Figure 4:
Figure 4:
Representative images of E. coli spheroplasts labeled with the membrane marker di-8-ANEPSS (red) interacting with FITC labeled BF2, Parasin, or hybrid analogs (green). Images are the middle z-stack of the cell shown at 63x magnification. Examples of a spheroplast showing both membrane translocation and membrane localizing are shown for each hybrid,
Figure 5.
Figure 5.
Relationship between membrane permeabilization of AMPs and antimicrobial activity (A) or membrane translocation (B). Membrane permeabilization was measured as the average relative fluorescence increase of propidium iodide five minutes after peptide addition to an E. coli culture (A and B). Membrane translocation is shown as the average percentage of E. coli spheroplasts to show peptide entry in confocal microscopy measurements (A). Antibacterial activity was measured against E. coli using a radial diffusion assay (B). Error bars represent the standard error of the mean.
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