Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.

Amphiphilic alpha-helices are common motifs used in numerous biological systems including membrane channels/pores and antimicrobial peptides (AMPs), and binding proteins, and a variety of synthetic biomaterials. Previously, an amphiphilic peptide with lysine-containing motifs was shown to reversibly bind the anionic porphyrin meso-Tetra(4-sulfonatophenyl)porphyrin (TPPS42−) and promote the formation of excitonically coupled conductive J-aggregates. The work presented here focuses on the use of this amphiphilic peptide and derivatives as a potential antimicrobial agent. AMPs are naturally occurring components of the innate immune system, which selectively target and kill bacteria. Sequence derivatives were synthesized in which the position of the Trp, used as a fluorescence reporter, was changed. Additional variants were synthesized where the hydrophobic amino acids were replaced with Ala to reduce net hydrophobicity or where the cationic Lys residues were replaced with diaminopropionic acid (Dap). All peptide sequences retained the ability to bind TPPS42− and promote the formation of J-aggregates. The peptides all exhibited a preference for binding anionic lipid vesicles compared to zwitterionic bilayers. The Trp position did not impact antimicrobial activity, but the substituted peptides exhibited markedly lower efficacy. The Dap-containing peptide was only active against E. coli and P. aeruginosa, while the Ala-substituted peptide was inactive at the concentrations tested. This trend was also evident in bacterial membrane permeabilization. The results indicate that the amphiphilic porphyrin binding peptides can also be used as antimicrobial peptides. The cationic nature is a driver in binding to lipid bilayers, but the overall hydrophobicity is important for antimicrobial activity and membrane disruption.

The continued emergence of new antibiotic resistant bacterial strains has resulted in great interest in the development of new antimicrobial treatments. Antimicrobial peptides (AMPs) are one of many potential classes of molecules to help meet this emerging need. AMPs are naturally derived sequences, which act as part of the innate immune system of organisms ranging from insects through humans. We investigated the antimicrobial peptide AP3, which is originally isolated from the winter flounder Pleuronectes americanus. This peptide is of specific interest because it does not exhibit the canonical facially amphiphilic orientation of side chains when in a helical orientation. Different analogs of AP3 were synthesized in which length, charge identity, and Trp position were varied to investigate the sequence-structure and activity relationship. We performed biophysical and microbiological characterization using fluorescence spectroscopy, CD spectroscopy, vesicle leakage assays, bacterial membrane permeabilization assays, and minimal inhibitory concentration (MIC) assays. Fluorescence spectroscopy showed that the peptides bind to lipid bilayers to similar extents, while CD spectra show the peptides adopt helical conformations. All five peptides tested in this study exhibited binding to model lipid membranes, while the truncated peptides showed no measurable antimicrobial activity. The most active peptide proved to be the parent peptide AP3 with the highest degree of leakage and bacterial membrane permeabilization. Moreover, it was found that the ability to permeabilize model and bacterial membranes correlated most closely with the ability to predict antimicrobial activity.

that the antimicrobial activity of these peptides can be correlated to the 3D-hydrophobic moment and to a simple structure-based packing parameter. This suggests that, in principle, one could design antimicrobial peptides based on such parameters. Our study shows that the nature of histidine favors its interaction with anionic lipid headgroups, i.e., a location at one end of an AMP, instead of the middle, and enhances the aggregation of cationic AMPs around anionic lipids, leading to transmembrane pore formation. The latter mechanism of disruption of the membrane can be correlated with the increased antimicrobial activity of these AMPs. Hence, the position of the histidine within the peptide sequence can be linked with AMP's mechanism of interaction with the membrane surface. Furthermore, the presence of histidine residue reduced the cytotoxic and hemolytic activity of the peptides, in some cases maintaining the same efficacy against bacteria. Some of these peptides have the potential to become good candidates to fight against bacteria. Acknowledgments: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNDI-UEFISCDI, project number PNII-123/2012, PNII-98/2012, PN-II-ID-PCCE-2011-2-0027, PN 09370301 and PN-II-RU-TE-2014-4-2418. Antimicrobial peptides (AMPs) are cationic, amphipathic proteins with an innate ability to kill a wide variety of pathogens, including viruses, fungi and bacteria. AMPs can be grouped into two general categories based on their mechanism of action. One group acts via permeabilization, or disruption of the cell membrane. The other group acts via translocation, or diffusion across the cell membrane and disruption of an intracellular process. Confocal microscopy is a technique readily used to identify AMP methods of action as it allows researchers to visualize peptide localization in bacteria by taking cross sections of cells. However, the small size and different orientations of bacteria can produce low-resolution images. To combat this problem our lab has used the cell-wall deficient spheroplast form of Escherichia coli to obtain higher quality images. Thesespheroplasts are both spherical and larger than typical E. coli, leading to improved imaging. Previously, we showed that several previously characterized AMPs have the same behavior against E. coli spheroplasts as normal cells. This project is an extension of that work aimed at developing protocols to consider the visualization of AMPs with cell-wall deficient forms of other bacterial strains. Characterizing AMPs against a variety of bacteria is important as research shows that AMPs antimicrobial properties differ against different bacteria strains. To this end, we are developing protocols to form protoplasts of the gram-positive bacteria Bacillus subtilis and Bacillus megaterium for imaging with well-characterized control peptides, such as buforin II and magainin. We have also investigated the membrane integrity of cell-wall deficient bacteria using a microscopy-based permeabilization assay. 1...