Revealing the Mode of Action of Halictine Antimicrobial Peptides: A Comprehensive Study with Model Membranes.

Antimicrobial peptides are innate host defense molecules with the ability to kill pathogens. They have been widely studied for their membrane lytic activity and their potential to overcome the ever-increasing threat of antimicrobial resistance against conventional antibiotics. Here, we focus on two halictines, antimicrobial peptides first obtained from the venom of the eusocial bee Halictus sexcinctus. The peptides, HAL-1 and HAL-2, are cationic (with +3 and +4 charges, respectively) and amphipathic, have 12 amino acid residues, and exhibit high biological activity. For this study, the mechanism of action of HAL-1 and HAL-2 was studied in detail using large and giant unilamellar vesicles composed of pure palmitoyl oleoyl phosphatidyl choline (POPC) and a mixture of POPC and the anionic lipid palmitoyl oleoyl phosphatidyl glycerol (POPG) as biomimetic models of the membranes of eukaryotes and microorganisms, respectively. A set of complementary techniques was put forward: carboxyfluorescein leakage assay, phase contrast optical microscopy, ζ-potential, static and dynamic light scattering, fluorescence and circular dichroism spectroscopies, and isothermal titration calorimetry. The results show that both halictines are able to interact strongly with anionic membranes: The interaction is exothermic and accompanied by structuring of the peptides as an α-helix and deep insertion into the membrane causing substantial membrane permeabilization at very low peptide/lipid molar ratios. Extensive vesicle aggregation was detected only at a high peptide concentration. On the other hand, the interaction of the halictines with POPC is significantly milder. Yet, the peptides were able to permeabilize the POPC membranes to some extent. Comparing both peptides, HAL-1 showed a somewhat stronger effect on model membranes. Fits to the data revealed apparent binding constants on the order of 103-104 M-1 for anionic membranes and 1 order of magnitude lower for zwitterionic bilayers. When lytic activity results were compared at the same bound peptide/lipid ratio, the halictines exhibited a higher activity toward zwitterionic membranes. As novel peptides, small and with powerful activity, these halictines are potential candidates for becoming antimicrobial agents.

Comparative study of the mechanism of action of the antimicrobial peptide gomesin and its linear analogue: The role of the ß-hairpin structure.

Gomesin (Gm) is an antimicrobial peptide first isolated from the hemolymph of a Brazilian spider. Its powerful antimicrobial activity is, however, accompanied by hemolysis. As an alternative to this issue, a linear analogue (named GmL) lacking the disulfide bonds was designed. Here, CD spectroscopy, a fluorescence-based leakage assay, isothermal titration calorimetry (ITC) and light scattering are used to study the interaction of both Gm and GmL with large unilamellar vesicles (LUVs) composed of POPC (palmitoyl oleoyl phosphatidylcholine) with 25 and 50 mol% POPG (palmitoyl oleoyl phosphatidylglycerol). The activities of Gm and GmL in respect to their binding affinity/enthalpy, ability to permeabilize membranes and to induce vesicle aggregation are correlated with peptide secondary structure. Whereas Gm displays a quite stable ß-hairpin motif irrespective of the environment, GmL assumes a random conformation in aqueous solution and in the presence of 25 mol% POPG but adopts a ß-like structure in the presence of 50 mol% POPG. Gm exhibited high lytic activity against both surface charge densities. Instead, the activity of GmL was found to be negligible in the presence of 25 mol% POPG LUVs, but comparable to that of the native peptide against 50 mol% POPG as a consequence of peptide structuring. We conclude that the activity of Gm and its linear analogue is intimately related to the formation of a ß-turn motif, in which the hydrophobic residues form a hydrophobic face able to insert into the membrane and disrupt it.

Structure-activity relationship of Trp-containing analogs of the antimicrobial peptide gomesin.

Gomesin (Gm) has a broad antimicrobial activity making it of great interest for development of drugs. In this study, we analyzed three Gm analogs, [Trp(1) ]-Gm, [Trp(7) ]-Gm, and [Trp(9) ]-Gm, in an attempt to gain insight into the contributions of different regions of the peptide sequence to its activity. The incorporation of the tryptophan residue in different positions has no effect on the antimicrobial and hemolytic activities of the Gm analogs in relation to Gm. Spectroscopic studies (circular dichroism, fluorescence and absorbance) of Gm and its analogs were performed in the presence of SDS, below and above its critical micelle concentration (CMC) (~8 mM), in order to monitor structural changes induced by the interaction with this anionic surfactant (0-15 mM). Interestingly, we found that the analogs interact more strongly with SDS at low concentrations (0.3-6.0 mM) than close to or above its CMC. This suggests that SDS monomers are able to cover the whole peptide, forming large detergent-peptide aggregates. On the other hand, the peptides interact differently with SDS micelles, inserting partially into the micelle core. Among the peptides, Trp in position 1 becomes more motionally-restricted in the presence of SDS, probably because this residue is located at the N-terminal region, which presents higher conformational freedom to interact stronger with SDS molecules. Trp residues in positions 7 and 9, close to and in the region of the turn of the molecule, respectively, induced a more constrained structure and the compounds cannot insert deeper into the micelle core or be completely buried by SDS monomers.

Resistance to degradation and cellular distribution are important features for the antitumor activity of gomesin.

Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15)]-Gm, and [Ser(2,6,11,15)]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15)]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15), Pro(9)]-D-Gm, and [Thr(2,6,11,15), D-Pro(9)]-Gm), which induced a similar ß-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. ß-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

Interaction of the antimicrobial peptide gomesin with model membranes: a calorimetric study.

Gomesin is a potent cationic antimicrobial peptide (z = +6) isolated from the Brazilian spider Acanthoscurria gomesiana . The interaction of gomesin with large unilamellar vesicles composed of a 1:1 mixture of zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and anionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) phospholipids is studied with isothermal titration calorimetry (ITC). In parallel, light scattering and optical microscopy are used to assess peptide-induced vesicle aggregation. The ability of gomesin to permeabilize the membrane is examined with fluorescence spectroscopy of the leakage of 5,6-carboxyfluorescein (CF). Vesicles coated with 3 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PE-PEG) lipids are also investigated to assess the influence of peptide-induced vesicle aggregation in the activity of gomesin. The ITC and light scattering titrations are done in two ways: lipid into peptide and peptide into lipid injections. Although some differences arise between the two setups, the basic interaction of gomesin with anionic vesicles is preserved. A surface partition model combined with the Gouy-Chapman theory is put forward to fit the ITC results. The intrinsic binding constant of gomesin is found to be K ≈ 10(3) M(-1). The interaction of gomesin with anionic membranes is highly exothermic and enthalpy-driven. Binding of gomesin is virtually always accompanied by vesicle aggregation and changes in membrane permeability, leading to CF leakage. Addition of PE-PEG to the membrane strongly attenuates vesicle aggregation but does not significantly change the mode of action of gomesin. The results point to a strong interaction of gomesin with the membrane surface, causing membrane rupture without a deep penetration into the bilayer core.

Cell-permeable gomesin peptide promotes cell death by intracellular Ca(2+) overload.

In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca(2+) signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 µM Gm increases the cytosolic Ca(2+) and induces membrane permeabilization after 30 min of treatment. Direct Ca(2+) measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca(2+) depletion, which in turn resulted in oscillatory mitochondrial Ca(2+) signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca(2+)-dependent pathway involving Gm translocation into the cell, ER Ca(2+) depletion and disruption, mitochondrial Ca(2+) overload and oxidative stress.

Revealing the lytic mechanism of the antimicrobial peptide gomesin by observing giant unilamellar vesicles.

Gomesin (Gm) is a potent cationic antimicrobial peptide from a Brazilian spider. Here we use optical and fluorescence microscopy to study the interaction of Gm, its low active linear analogue, [Ser(2,6,11,15)]-Gm (GmL), and a fluorescent labeled analogue, Gm-Rh, with giant unilamellar vesicles (GUVs) composed of mixtures of the neutral lipid palmitoyloleoyl phosphatidylcholine (POPC) with the negatively charged lipid palmitoyloleoyl phosphatidylglycerol (POPG) or cholesterol, so as to mimic bacterial and mammalian cell membranes, respectively. We observed the effect of injecting a peptide solution with a micropipet close to GUVs. As a result of peptide-lipid interaction, GUVs burst suddenly. Stable pores, which result in leaky vesicles, were not observed. Fluorescence microscopy of Gm-Rh injected on GUVs confirmed the high peptide/lipid affinity. These facts lead us to suggest that Gm and GmL disrupt the membrane via the carpet model. In order to quantify the lytic activity of both peptides against different membrane composition, a solution of GUVs was diluted in increasing concentration of peptides and the fraction of burst GUVs was measured as a function of time. The lytic activity of both peptides was enhanced by the presence of POPG and decreased upon addition of cholesterol. GmL exhibited lower lytic activity as compared to Gm, but this difference vanished at high POPG molar fraction.