Introduction

The increasing emergence and dissemination of antibiotic resistance among bacterial pathogens has become a global public health challenge1,2. Therefore, there is an urgent need to develop new antimicrobial agents to overcome this problem. Antimicrobial peptides (AMPs) are an essential component of the innate immune system produced as a first line of defense by all multicellular organisms3. With such exceptional properties as broad-spectrum antimicrobial activity, rapid action and infrequent development of resistance4,5, AMP-based pharmaceuticals provide excellent templates for a wide range of antimicrobial agents and biomedical applications.

In general, naturally occurring AMPs are between 12 and 50 amino acids in length, and often contain cationic and hydrophobic residues6. Previous studies contributing to the understanding of the structure-activity relationship of AMPs show that one important class of membrane-active AMPs infers an amphipathic α-helical conformation4,7,8. The initial electrostatic interaction between a positively-charged AMP and the negatively-charged microbial cell membrane, such as lipoteichoic acids in Gram-positive bacteria or lipopolysaccharides (LPSs) in Gram-negative bacteria, results in insertion of a hydrophobic segment into the lipid bilayer of the microbial membranes. The effect is to destabilize by pore formation and produce cell death, a process unlike that of conventional antibiotics which have specific modes of action such as inhibition of cell wall or nucleic acid synthesis9.

Cathelicidins, a prominent family of AMPs, play important roles in the innate immune system of practically all species of vertebrates10. The α-helical cathelicidins possess broad‐spectrum antimicrobial activity against bacteria, fungi and viruses, and of note also against antibiotic-resistant strains of pathogenic bacteria11. Besides their antimicrobial activity, cathelicidins bind and neutralize LPS, and protect against endotoxic shock in a murine model of septicemia12. Frog skin is a natural storehouse of active AMPs13. Upon contact with microorganisms, anuran skin peptides are produced in dermal serous glands and stored within granules for release onto the skin surface after stress or tissue injury as part of the immune system14. Amongst them, aurein peptides from the Australian southern bell frogs Litoria aurea and Litoria raniformis15, are a large family of peptides with prominent activity toward both bacteria and cancer cells16.

Despite the many desirable properties of AMPs, they also exhibit undesirable properties including hemolytic activity toward human red blood cells, sensitivity to protease, salt and serum, and high production cost which impede their development as therapeutic agents17. However, optimization of sequences or modification of AMPs can improve antimicrobial effects while reducing cytotoxicity and so overcome barriers and expedite development for clinical application7,18,8. The truncation of LL-37, based on the amino acid composition and 3D structure, retains its antimicrobial activity while losing its side effects21. The synthetic hybrid peptides of progetrin-1, bovine lactoferricin and cecropin A, LB-PG and CA-PG, exhibit broad antimicrobial activity against both Gram-positive and -negative bacteria along with reduced hemolytic activity8,27,38. We therefore modified parent peptide sequences by truncation of unstructured regions and amino acid substitution. Their derivatives, especially P7, showed perfect amphipathicity and higher hydrophobicity than the parental peptides with significantly improved antimicrobial activity. These results suggested that the unstructured region at positions 1 to 3 did not affect antimicrobial activity, but that both amphipathicity and hydrophobicity played crucial roles in antimicrobial activity. In addition, the potent antimicrobial activity of P7 may have resulted from the bulky side chain of tryptophan at position 7. Since this located on the hydrophobic/hydrophilic interface, the interaction of peptides with bacterial membranes is increased8.

Combinations of two or more native peptides, or hybrid peptide derivatives, have received great attention, as this can enhance their antimicrobial properties22. Since P7 showed much higher antimicrobial activity than A3, the sequence of P7 was maintained with its activity further improved by hybridizing with A3 at the C-terminus. P7A3 displayed higher antimicrobial activity against all tested bacteria when compared with P7 and A3 alone, however P7A3 also displayed the strongest hemolytic activity. Our results indicated that the high positive charge and hydrophobicity of P7A3 correlated with both its antimicrobial and hemolytic activities. To minimize toxicity, truncated derivatives were designed to identify the shortest amino acid sequence which retained the potent antimicrobial activity while reducing toxicity. It has been shown that the N-terminal α-helical domain of peptides such as PMAP-36 is its active region and has the ability to interact with and penetrate the bacterial membranes8. Moreover, an unstructured region (position 18 to 23) was observed in the C-terminus of the ribbon structure of P7A3. Therefore, the C-terminus of the hybrid peptide was truncated. The truncated derivatives of the hybrid analogue (PA-14 and -13) displayed high antimicrobial activity against both Gram-negative and -positive bacterial strains with relatively decreased toxicity. Our studies indicated that increasing amphipathicity (with suitable positive charges) and hydrophobicity improved antimicrobial activity and selectivity. Among truncated derivatives, the shortest peptide (PA-13) showed potent antimicrobial activity along with low toxicity against both hRBCs and L929 cells. Differences in amino acid sequence and position have been suggested to be important for antimicrobial activity. Importantly, the bulky side chain of tryptophan at the hydrophobic/hydrophilic interface of all truncated derivatives played an important role in facilitating the interaction of peptides with bacterial membranes. The antimicrobial activity and toxicity of AMPs are related to multiple physio-chemical properties, for instance the length, charge, hydrophobicity, amphipathicity and hydrophobic/hydrophilic angle. Thus, adjusting one can cause alterations to the others and so it is difficult to highlight the influence of a single factor for the activity4.

The therapeutic index is a widely employed parameter to indicate the specificity of AMPs. It is calculated as the ratio of MHC (hemolytic activity) and MIC (antimicrobial activity)27. Thus, larger TI values indicate greater antimicrobial specificity. Among all derivative peptides, P7 exhibited the highest TIs (8.78 and 2.06 against Gram-negative and -positive bacteria, respectively). The hybrid peptide P7A3 displayed high antimicrobial activity coupled with strong hemolytic activity. Hence its TI was low (0.09 and 0.31 for Gram-negative and -positive bacteria, respectively). PA-18, a truncated form containing the entire α-helical structure of the hybrid peptide, displayed comparable specificity with P7A3. Among truncated derivatives, PA-13 exhibited the highest TI against all tested bacterial strains suggesting that it had increased selectivity toward bacterial cells compared with hRBCs, and implying it had a wider therapeutic window. Our studies demonstrated that an increase of TI mainly resulted from a reduction of hemolytic activity, and indicated that truncation at the C-terminus of peptides affected their hemolytic activity. Therefore, the potent antimicrobial activity with low host toxicity of PA-13 reflects an attractive direction for AMP development.

It has been suggested that the propensity of AMPs to form an amphipathic α-helix in membrane-mimetic environments is the key to their membrane-disruptive activity27. In the current study, the secondary structure of PA-13 in water, membrane-mimicking environments and LPS was analyzed using CD spectra. The results indicated that PA-13 exhibited a random coil in aqueous environments. In 30 mM SDS, 50% TFE and 0.05% LPS of P. aeruginosa, PA-13 exhibited a typical α-helical structure, with more helical content in 30 mM SDS than in 50% TFE and 0.05% LPS. This result was consistent with previous studies indicating that the structural change of peptides from a random coil to an α-helix structure might be one of the most important factors determining peptide function at bacterial cell membranes, including antimicrobial activity39.

Previous studies have demonstrated that a positive charge facilitates antimicrobial peptide binding to the negatively-charged bacterial membranes, and/or LPS, via electrostatic interactions40. However, other positively charged molecules, including salts, can weaken this electrostatic interaction. The antibacterial activity of PA-13 in the presence of different physiological salts was investigated. PA-13 retained its antimicrobial activity in the presence of NH4+, Zn2+, Fe3+, Ca2+ and K+, suggesting that cation valence [monovalent (NH+, K+), divalent (Zn2+, Ca2+) and trivalent (Fe3+)] had little or no effect on the strength of PA-13’s antimicrobial activity. This might be due to the bulky side chain of tryptophan, which could enhance the affinity of this antimicrobial peptide for the bacterial membrane and contribute to its strong antibacterial activity in the presence of salts8. However, there was a marked decrease in the antibacterial activity of PA-13 against P. aeruginosa in the presence of Na+ and Mg2+. Mg2+ compromised the antimicrobial activity, possibly due to competition with cationic peptide for binding with LPS molecules on bacterial outer membranes. The presence of Na+ could hinder electrostatic interactions and decrease the binding efficacy of the peptide. This might be attributed to the first step in antimicrobial activity, ultimately compromising the killing efficiency of a peptide41,42. However, this is in consistent with the results of other studies43,44. While F4 peptide retains killing activity in the presence of salts at physiologic concentrations, except Na+ and Mg2+, it still displays effective antimicrobial potency in the mouse model45.

Although the exact mechanism of action of AMPs has not been established, it has been proposed that the cytoplasmic membranes of bacteria are the main target of these peptides via their disruption or the formation of pore/ion channels35. To assess membrane-penetrating activity and localization, TAMRA-labelled PA-13 was incubated with P. aeruginosa and the bacteria investigated by flow cytometry and confocal microscopy. The results indicated that PA-13 exhibited time-dependent penetrating activity. PA-13 rapidly punctured the bacterial membranes (within 5 min at 1 × MIC). The results were strongly in accordance with localization studies. That is, TAMRA-labelled PA-13 clearly localized on P. aeruginosa membranes and then accumulated in bacterial cytoplasm. After inserting into the cytoplasmic membrane, PA-13 potentially disrupted the membrane integrity in dose- and time-dependent manner. This appeared concordant with the collapse of membrane electrical potential as observed by flow cytometry. The results of SEM and TEM further confirmed the killing mechanisms of PA-13 being membrane rupture and pore formation, leading to the leakage of intracellular contents and cell death. The fluorophore-PEN conjugates displayed altered modes of membrane interaction with increased insertion into the core of the model cell membrane and exert membrane-thinning effects without effecting membrane-penetrating ability46. This agrees with our results, showing the penetrating ability of TAMRA-labelled PA-13 in a time- dependent manner and that the unlabeled PA-13 behaved similarly to the labeled compound determined by circular dichroism and flow cytometry (data not shown).

Lipopolysaccharide from bacterial endotoxin, a major component of the outer membrane of all Gram-negative bacteria, can cause inflammation, sepsis and shock47. Previous research demonstrated that the peptide-LPS interaction strongly promotes bacterial cell death and reduces inflammation. Anti-inflammatory activity of AMPs can occur as a result of neutralization of biological inflammatory cytokines or inhibition of their production48. Our studies revealed that PA-13 was able to bind and neutralize LPS, the negatively charged molecules constituting an important barrier in the membranes of Gram-negative bacteria, in a dose-dependent manner. Furthermore, PA-13 showed dose-dependent inhibition of LPS-mediated, Toll-like receptor activation, which inhibited the release of pro-inflammatory cytokines and down-regulated further severe inflammation. Therefore, PA-13 appears to be a very promising anti-inflammatory agent.

There are several reports on the relationship between in vitro activity and in vivo efficacy and safety of antimicrobial peptides49,50,51,52,53,54. A novel peptide, BSN-37, exhibits strong antibacterial activity against S. Typhimurium and significantly inhibits the proliferation of intracellular S. Typhimurium with no associated toxicity to the eukaryotic cells49. A broad-spectrum antimicrobial peptide, GL13K, with low cytotoxicity in vitro exhibits a low level of hemolysis and anti-inflammatory activity in vivo in a mouse model of inactivated LPS-induced sepsis50,51. Although less peptide binding is observed, GL13K is still active in physiological salts conditions52. The in vivo protective effect of buforin II in an experimental rat model of A. baumannii sepsis confirms the in vitro effectiveness of this peptide53. The proline-rich, bovine innate immune peptide, Bac5, is able to kill some mycobacterial species and supports macrophage activation in vitro in synergy with Mycobacterium marinum. Peptide Bac5 is able to slow M. marinum infections of zebrafish54. The in vitro characterization of antimicrobial peptides may be applicable as predictors of in vivo efficacy and safety. Our in vitro data may, similarly, predict the in vivo efficacy and safety of PA-13. To continue the clinical development of this peptide, studies of efficacy and safety in animal models will be conducted.

In conclusion, among rationally designed, short α-helical hybrid peptides inspired by cathelicidin and aurein, PA-13 exhibited potent antibacterial activity against P. aeruginosa, including multidrug resistant strains, via penetration through the bacteria’s membrane and causing depolarization, permeabilization and rupture of membranes, and subsequent leakage of intracellular contents and cell death. The peptide displayed low hemolytic and cytotoxic activities against mammalian cells suggesting a therapeutic potential. Furthermore, PA-13 showed anti-inflammatory activity via LPS neutralization. Our research supports the usefulness of rational design of hybrid peptides as promising candidates for development of novel antimicrobial agents.