In a recent study published in NPJ Antimicrobials and Resistance, a gaggle of researchers investigated the synergistic motion of six winter flounder (WF) antimicrobial peptides (AMPs) against bacterial pathogens, uncovering the therapeutic potential of mixing potent and weakly lively peptides.

Study: Synergy between Winter Flounder antimicrobial peptides. Image Credit: MicheleUrsi/Shutterstock.com

Background

Although AMPs offer a promising alternative to traditional antibiotics, and a couple of exhibit strong bactericidal properties, the role of less lively ones stays ambiguous.

For instance, the WF produces pleurocidin, an AMP with membrane-damaging and intracellular targeting abilities that would revolutionize therapeutic approaches and counteract antibiotic resistance. Nevertheless, its interactions and effects on bacterial elimination warrant further investigation.

Concerning the study

In the current study, peptides were sourced from Cambridge Research Biochemicals, and further refined with water/acetonitrile gradients. Lipids got here from Avanti Polar Lipids, Inc. and were used unpurified.

To check the antibacterial properties, a modified broth microdilution method was employed. The minimum inhibitory concentration (MIC) was assessed using a Clariostar plate reader.

Tekin et al.’s method, was used to measure antibacterial activity in triplicate. Peptide concentrations inhibiting 10% of bacterial growth were utilized in synergy screens. Combos of peptides were evaluated through an adjusted MIC method.

The Fractional Inhibitory Concentration (FIC) was determined, with particular values indicating synergistic effects. Standard microdilution Chequerboard assays measured synergy, defining growth/no growth and calculating the FIC.

Using multiple antibiotic and bacterial strains, the in vitro pharmacodynamic assays observed bacterial behavior under various antimicrobial exposure. The bacterial growth rate under the antimicrobial influence was plotted and analyzed. Galleria mellonella larvae tested the antimicrobial efficiency of assorted treatments on a burn wound infection model.

Nuclear magnetic resonance (NMR) structure determination involved peptide samples mixed with specific chemical compounds, with NMR spectra obtained from Bruker spectrometers. The resulting data were processed and analyzed using particular software.

Molecular dynamics simulations were conducted using high-performance systems, analyzing peptides in lipid environments using the chemistry at Harvard macromolecular mechanics 36 (CHARMM36) force field. The peptide structures were introduced above lipid bilayers, with various aspects adjusted for accurate simulations.

The researchers used diphytanoyl chain lipids to create giant unilamellar vesicles (GUVs). These were formed utilizing the electroformation method. Bilayers were then made and tested with peptides, and data was recorded and analyzed.

Study results

In the current study, the researchers examined the antimicrobial activity of six WF AMPs on bacterial isolates. Two representative Vibrio isolates were included resulting from the marine origin of WF peptides. It was observed that WF2 (pleurocidin) and WF1a-1 showed high antimicrobial potency.

D-analogues were prepared to further analyze their activity against P. aeruginosa isolates. The findings showed D-WF1a-1’s performance closely matched or barely exceeded D-pleurocidin. Nevertheless, the antibacterial activity of WF4 was predominantly against Gram-negative bacteria, while WF1, WF1a, and WF3 showed minimal activity.

Three methods were employed to look at synergy amongst WF AMPs. Initially, the strategy of Tekin et al. was utilized, identifying synergistic two-way combos of several AMPs. When examining higher-order conditions, the synergy emerged from your complete combination reasonably than individual pairings.

Subsequently, researchers sought to discover probable synergistic binary combos for antibiotic-resistant isolates using two different screening methods. The outcomes showed nine of fifteen two-way WF AMP combos displayed strong synergy, depending on the bacterial isolate tested.

Notably, each WF AMP participated in no less than one synergistic combination. Lastly, researchers explored whether D-WF1a synergizes with D-pleurocidin or its analog, D-pleurocidin-KR.

Beyond merely considering gains in antibacterial potency, the researchers sought to know the behavioral changes in WF AMPs resulting from synergy. Various studies had noted increased cooperativity in bactericidal killing rates for multi-AMP combos.

The present study tested the in vitro desirable pharmacodynamic (PD) profile of WF2 against its synergistic partners. The findings demonstrated that each one WF AMPs and their combos were substantially more bactericidal than clinically used antibiotics, killing bacteria in minutes in comparison with antibiotics which took hours.

They didn’t test WF1a and WF3’s antibacterial potency individually. Nevertheless, combining them with pleurocidin/WF2 enhanced bactericidal cooperativity. This effect varied depending on bacterial resistance and the peptide enantiomer used.

The mix of WF AMPs showed a major enhancement of their therapeutic efficacy, in response to research conducted on an invertebrate burn wound infection model using Galleria mellonella larvae.

This model was employed to check the therapeutic performance of assorted antibiotic combos. Among the many antibiotics tested, gentamicin demonstrated strong protective effects as a standalone treatment, outperforming other antibiotics like ciprofloxacin, imipenem, and meropenem.

Nevertheless, an intermediate dose of WF2, often called pleurocidin, was just as effective as gentamicin at a particular concentration. Interestingly, the combined low doses of WF2 with either WF1a or WF3 resulted in protection levels much like gentamicin, although these peptides provided no protection individually.

A mixture of WF3 and WF4 also showcased complete protection, contrasting their ineffectiveness when used alone.

The research further delved into the molecular intricacies behind the synergistic effect of those WF AMPs. It was observed that although certain peptides shared sequence similarities, their antibacterial properties and synergistic potential differed markedly.

This led to the hypothesis that the mode of motion might involve the formation of aggregates or hetero-oligomers, on condition that single AMPs cannot form pores in lipid membranes individually.

The structural characteristics of WF peptides were analyzed, revealing that they’ll adopt specific conformations, with some peptides exhibiting more hydrophobic or hydrophilic tendencies depending on the environmental pH.

One other significant finding was the conformational flexibility of those peptides, as they might assume each α-helix and PII conformations. These conformations were inferred through techniques like far-ultraviolet circular dichroism and NMR spectroscopy.

Moreover, molecular dynamics simulations provided insights into the peptides’ interaction with bacterial plasma membranes. Notably, differences in bilayer penetration and lipid chain disordering were identified among the many WF AMPs, with some showing deeper penetration in specific bilayer conditions.

When combined in synergistic pairs, these peptides exhibited altered penetration, acyl chain disordering, and peptide-lipid hydrogen bonding, highlighting the complex molecular interplay resulting in enhanced therapeutic outcomes.

Conclusion

The research explored the atomic interaction of WF AMP with membranes, aiming to relate it to bactericidal activity and understand its role in antimicrobial potency. Although pleurocidin can penetrate bacteria, its membrane disruption properties are noteworthy.

The team identified the minimum concentration of peptide-inducing ion conductance using patch-clamp experiments. Findings showed that while WF AMP’s ability to penetrate and disorder lipids varied, this didn’t directly relate to antibacterial strengths.

Different WF AMPs showed varied activities and effects on modeled bacterial membranes. Combining WF1a and WF2 led to enhanced ion conductance, suggesting a synergistic relationship.