High specific selectivity and Membrane-Active Mechanism of the synthetic centrosymmetric α-helical peptides with Gly-Gly pairs

The widespread increase in bacterial resistance to many conventional antibiotics has become a worldwide problem. Despite the abuse of antibiotics has been strictly restricted, the continuous increasing prevalence of multidrug-resistant bacteria still makes people highly nervous. Furthermore, the multidrug-resistant bacteria infections associated to medical implantation material are also threatening the human health, but for example, urinary catheter coatings are unable to provide sufficient protection against multidrug-resistant bacteria1. Therefore, the development of alternative antimicrobial coatings materials against multidrug-resistant bacteria is extremely urgent. One potential source of novel antibiotics is Gene-encoded antimicrobial peptides (AMPs), evolutionarily ancient weapons against invading microbes, which are the first barrier of the innate immune system in most multicellular organisms2. AMPs have been isolated and characterized from practically all living organisms, ranging from prokaryotes to humans3. Currently, the known primary structures of AMPs vary widely, and more than 2400 sequences have been documented in the Antimicrobial Peptide Database (APD) (http://aps.unmc.edu/AP/main.php). The most abundant type of peptide is the α-helical AMP, with over 340 being reported (see the APD), and they appear to represent particularly successful structural arrangements in innate defense4. They have broad-spectrum antibiotic activity, kill bacteria by interacting with the bacterial cell membranes and differ from antibiotics that target specific molecular receptors of pathogens5. Consequently, it will be difficult for bacteria to evolve resistance to α-helical AMPs unless bacteria alter their cell membrane composition6. Based on the above mechanism of action, α-helical AMPs are an ideal candidate for replacing conventional antibiotics and have potential as antimicrobial coating materials for controlling implant-related infections, which aid in the development of new bioengineering and biomedical materials7. Despite α-helical AMPs bring the hope and opportunity to overcome the drug-resistance bacteria, some shortcomings, such as systemic toxicity, ease of degradability, and high cost, still impede the further development of natural peptides as therapeutic drugs and peptide-based biomaterials. Various methods have been employed to modify and/or optimize the natural α-helical AMPs, which focus on improving antimicrobial activity, while reducing the undesirable cytotoxicity towards mammalian cells. But these methods are complicated, random and lack systematic design principles8. Moreover, some reports showed that widespread clinical use of AMPs with sequences that are too close to those of human AMPs would inevitably compromise own natural defenses9. In this view, synthetic AMPs are a viable alternative10. The template-assisted approach is a promising method to guide synthetic AMPs design, which maintains evolutionarily conserved sequence patterns and reduces the number of the candidate AMPs required to obtain useful results. The first typical sequence templates were proposed by Tossi et al.11, which were obtained by comparing and analyzing the sequences of over 150 naturally occurring α-helical peptides12. By further comparatively analyzing the typical sequence templates, we found an interesting phenomenon in that the sequences of amino acids were nearly a centrosymmetric distributed sequence (Supplementary Fig. S1A and S1B). For these reasons, we prudently hypothesized that the centrosymmetric sequence might be a relic of evolutionary divergence that contributed to the antimicrobial activity. Some literatures have demonstrated part of our conjecture that the symmetrical distribution of amino acids is a promising strategy for optimizing the natural β-turn peptides with strong antimicrobial activity7,13,14. While there is little research describing such design ideas for α-helical peptides, whether derivative from natural peptides or synthetic peptides. Most of all, they do not establish a systematical and universally applicable centrosymmetric sequence template to guide the AMPs design, whether α-helix or β-turn. Small amino acids (Ala and Gly) are always ignored or replaced in the modification and/or optimization process of the natural peptides15,16. However, some new ideas were put forward that the rational use of small amino acid substitutions can improve the biological properties of AMPs; in particular, selectivity (reduced cytotoxicity) is improved17,18. And the motifs of two small residues are present with high frequency in the transmembrane helical domains19. Therefore, In this study, we established a primary centrosymmetric α-helical sequence template based on the α-helical protein-folding principles to conserve amphipathicity, which is a necessary property of peptides interacting with target membranes20. The primary sequence of the centrosymmetric α-helical template is the (y + hhh + y)n amino acid sequence (h, hydrophobic amino acid; +, cationic amino acid; y, Gly or hydrophobic amino acid) and a series of centrosymmetric peptides was designed with the pairs of small amino acids (Ala and Gly). Furthermore, the corresponding peptides with scrambled centrosymmetric sequences were also designed. All the peptides were first characterized by circular dichroism (CD) for their secondary conformation in phosphate buffer solution (PBS) and in membrane-mimicking environments (sodium dodecyl sulfate (SDS) and trifluoroethyl alcohol (TFE)). Then, the hemolytic properties, cytotoxicity towards mouse macrophage RAW264.7 and antimicrobial activity of peptides were measured in vitro; the salt sensitivity of the peptides was determined against model microbes, such as Escherichia coli (E. coli) ATCC 25922 and Staphylococcus aureus (S. aureus) ATCC29213. The capability of the peptides to synergize with antibiotics was evaluated using the checkerboard titration method. Flow cytometry, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) allowed direct observation of the changes in cell morphology and the integrity of cell membranes after peptide treatment. Assays examining liposome leakage, outer membrane permeabilization and membrane depolarization were employed to investigate the membrane destruction mechanisms of the peptides, and DNA-binding assays were employed to investigate the cell death as well as the possible intracellular targets of the peptides.