Temporin H

Background: Temporin is one family of the shortest antimicrobial peptides found in Ranidae frogs. Staphylococcus aureus is one of the main pathogens of suppurative diseases and food contamination, causing severe local or systemic infections in humans. Temporin-GHa (GHa) was previously obtained from Hylarana guentheri, showing weak antibacterial activity against S. aureus. Most temporin peptides are positively charged by arginine and lysine; however, GHa contains histidine. Objective: In order to investigate the impact of positively charged amino acid on its antibacterial and antibiofilm activity, GHa4R was designed and synthesized by replacing histidine with arginine in GHa. Method: The antibacterial activity and efficacy against S. aureus were detected by minimum inhibitory concentration, minimum bactericidal concentration, and time-killing kinetics assays. The action mechanism was determined by propidium iodide uptake and scanning electron microscopy assays. The antibiofilm activity was measured by the MTT method. Eradication of biofilm was observed by fluorescence microscope. Results: Compared to GHa, GHa4R had stronger antibacterial activity and bactericidal efficacy against S. aureus. Impressively, GHa4R presented antibacterial activity against methicillin-resistant S. aureus (MRSA). It was barely affected by temperature, pH, and storage period, showing high stability. Furthermore, it increased the permeability of the cell membrane and damaged the membrane integrity, leading to cell death. In addition, GHa4R did not induce antibiotic resistance in S. aureus in 30 days, but the MIC of vancomycin was doubled. It not only inhibited S. aureus biofilm formation but also eradicated 24 h-biofilms. Conclusion: The above-mentioned characteristics make GHa4R a promising candidate for the treatment of S. aureus infections.

Several antimicrobial peptides (AMPs) have been reported in amphibian toxins, as temporin-PTa from Hylarana picturata. The amino acid distribution within a helical structure of AMPs favors the design of new bioactive peptides. Therefore, this work reports the rational design of two new synthetic peptides denominated Hp-MAP1 and Hp-MAP2 derived from temporin-PTa. These peptides present an amphipathic helix with positive charges of +4 and +5, hydrophobic moment () of 0.66 and 0.72 and hydrophobicity () of 0.49 and 0.41, respectively. Hp-MAP1 and Hp-MAP2 displayed in vitro activity against Gram-negative and Gram-positive bacteria from 2.8 to 92 µM, without presenting hemolytic effects. Molecular dynamics simulation suggested that the parent and designed temporin-like peptides lack structural stability in an aqueous solution. By contrast, α-helical structures were predicted in hydrophobic and anionic environments. Additionally, the peptides were simulated on mimetic membranes composed of anionic and neutral phospholipids 1,2-dipalmitoylsn-glycerol-3-phosphatidylglycerol (DPPG-anionic), 1,2-dipalmitoyl-sn-lyco-3 phosphatidylethanolamine (DPPE-neutral). When in contact with DPPG/DPPE (90:10) and DPPG/DPPE (50:50) temporin-PTa, Hp-MAP1 and Hp-MAP2 established interactions guided by hydrogen and saline bounds. Therefore, the findings described here reveal that the optimization of the amphipathic α-helical cationic peptides Hp-MAP1 and Hp-MAP2 enabled the generation of new synthetic antimicrobial agents to combat pathogenic microorganisms.

Few antifungal agents are currently available for the treatment of fungal infections. Antimicrobial peptides (AMPs), which are natural molecules involved in the innate immune response of many organisms, represent a promising research method because of their broad killing activity. The aim of this study was to assess the activity of a frog AMP, [K3]temporin-SHa, against some species of yeasts and moulds, and to further explore its activity against Candida albicans. MIC determinations were performed according to EUCAST guidelines. Next, the activity of [K3]temporin-SHa against C. albicans was explored using time-killing curve experiments, membrane permeabilization assays, and electron microscopy. Finally, chequerboard assays were performed to evaluate the synergy between [K3]temporin-SHa and amphotericin B or fluconazole. [K3]temporin-SHa was found to be active in vitro against several yeasts with MIC between 5.5 and 45 µM. [K3]temporin-SHa displayed rapid fungicidal activity against C. albicans (inoculum was divided into two in less than an hour and no viable colonies were recovered after 5 h) with a mechanism that could be due to membrane permeabilization. [K3]temporin-SHa was synergistic with amphotericin B against C. albicans (FICI = 0.303). [K3]temporin-SHa could represent an additional tool to treat several Candida species and C. neoformans.