Temporin-PTa

Bacterial and fungal infections are challenging due to their low susceptibility and resistance to antimicrobial drugs. For this reason, antimicrobial peptides (AMP) emerge as excellent alternatives to overcome these problems. At the same time, their active insertion into the cell wall of microorganisms can be availed for biorecognition applications in biosensing platforms. Temporin-PTA (T-PTA) is an AMP found in the skin secretions of the Malaysian fire frog Hylarana picturata, which presents antibacterial activity against MRSA, Escherichia coli, and Bacillus subtilis. In this work, T-PTA was explored as an innovative sensing layer aiming for the electrochemical differentiation of Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus subtilis, Enterococcus faecalis, Candida albicans, and C. tropicalis based on the structural differences of their membranes. The biosensor was analyzed through electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). In this approach, the different structural features of each microorganism resulted in different adherence degrees and, therefore, different electrochemical responses. The transducing layer was fabricated by the self-assembling of a 4-mercaptobenzoic acid (MBA) monolayer and gold-capped magnetic nanoparticles (Fe3O4@Au) implemented to improve the electrical signal of the biointeraction. We found that each interaction, expressed in variations of electron transfer resistance and anodic peak current, demonstrated a singular response from which the platform can discriminate all different microorganisms. We found expressive sensitivity towards Gram-negative species, especially K. pneumoniae. A detection limit of 101 CFU.mL-1 and a linear range of 101 to 105 CFU.mL-1 were obtained. The T-PTA biosensor platform is a promising and effective tool for microbial identification.

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.

A new approach is applied to the design and study of the antibacterial activity of novel Temporin-Pta peptides. Using Temporin-Pta as a template, together with spiral structure domain breaking and amino acid residue substitution principles, antibacterial peptides are reformed to create new antibacterial peptides. The broth dilution analysis method was used to determine the bacteriostatic effect of the new Temporin-Pta structures, and the hemolytic effect and protease stability of the new peptides were studied. Results showed that the modified antibacterial peptide HX-12A has higher inhibitory effects of active protease stability on E. coli and other bacteria and the hemolytic ability is below 5%, thus achieving successful new Temporin-Pta transformations. It is also shown that the corresponding bacteriostatic effects of these antibacterial peptides are closely related to the differences in structure and composition. This method can improve the biological activity of the antibacterial peptide and drug resistance that is normally difficult to achieve. The present study provides a good theoretical basis for further research on antimicrobial peptide structures and functions.