Development of a Novel Short Synthetic Antibacterial Peptide Derived from the Swallowtail Butterfly Papilio xuthus Larvae

Insects possess biological defense systems that can effectively combat the invasion of external microorganisms and viruses, thereby supporting their survival in diverse environments. Antimicrobial peptides (AMPs) represent a fast-acting weapon against invading pathogens, including various bacterial or fungal strains. A 37-residue antimicrobial peptide, papiliocin, derived from the swallowtail butterfly Papilio xuthus larvae, showed significant antimicrobial activities against several human pathogenic bacterial and fungal strains. Jelleines, isolated as novel antibacterial peptides from the Royal Jelly (RJ) of bees, exhibit broad-spectrum protection against microbial infections. In this study, we developed a novel antimicrobial peptide, PAJE (RWKIFKKPFKISIHL-NH2), which is a hybrid peptide prepared by combining 1–7 amino acid residues (RWKIFKK-NH2) of papiliocin and 1–8 amino acid residues (PFKISIHL-NH2) of Jelleine-1 to alter length, charge distribution, net charge, volume, amphipaticity, and improve bacterial membrane interactions. This novel peptide exhibited increased hydrophobicity and net positive charge for binding effectively to the negatively charged membrane. PAJE demonstrated antimicrobial activity against both gram-negative and gram-positive bacteria, with very low toxicity to eukaryotic cells and an inexpensive process of synthesis. Collectively, these findings suggest that this novel peptide possesses great potential as an antimicrobial agent.

isolated a novel antibacterial peptide, Jelleine, from the Royal Jelly (RJ) of bees and reported its properties [1]. Jelleines are very short peptides, consisting of 8 to 9 amino acid residues, produced constitutively to provide broad-spectrum protection against microbial infections [1]. The Jelleines present a hypothetical net charge of 1+ or 2+, and most of these residues are hydrophobic [1]. Therefore, Jelleines represent the basic structural properties of the antimicrobial peptides.
It is essential to study the properties of peptides to develop new antimicrobial agents for both, gram-negative and gram-positive bacteria. AMPs play essential roles in peptide interactions with the membrane surface and/or the membrane core. Thus, the design of potent peptides have to take into consideration factors such as length, charge distribution, net charge, volume, amphipaticity, and oligomeric state in solution [1]. In this study, we designed and synthesized a novel hybrid peptide, PAJE (RWKIFKKPFKISIHL-NH 2 ), composed of 1-7 Nterminal amino acid residues of papiliocin (RWKIFKK-NH 2 ) followed by 1-8 N-terminal residues of Jelleine-1 (PFKISIHL-NH 2 ) to develop a potent antibacterial peptide with a broad-spectrum of activity against grampositive and gram-negative bacteria. N-terminal helix residues of papiliocin are essential for the rapid permeabilization of the gram-negative bacterial membrane [6]. Our results suggest that a novel 15-mer non-toxic antimicrobial peptide is highly effective against gram-negative and gram-positive bacteria.
The antibacterial activity of synthetic PAJE was examined by the agar well diffusion assay. Briefly, single colonies of bacteria were inoculated in tryptic soy broth (TSB; Difco, USA) and cultured overnight at 37°C. The cultures were subsequently diluted in fresh TSB and incubated until the optical density at 600 nm reached 0.4. A volume containing 4 × 10 6 CFU bacteria was inoculated into 10 ml of worm citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) containing 1% low electroendosmosis-type agarose (Sigma-Aldrich, USA) and 0.03% TSB. The mixture was rapidly poured into a sterile petri-dish to form a uniform layer, after which 3-mm-diameter holes were punched in the set agarose and filled with 10 μl of test peptides. After allowing 3 h for diffusion of the samples, 10 ml of TSB medium containing 1% agar was overlaid and incubated overnight at 37°C. Activity of the synthetic peptide was measured from its inhibitory zone.
In addition to the agar well diffusion assay, the MIC (minimum inhibitory concentration) of synthetic PAJE was also determined using the broth microdilution assay and compared with those of Jelleine-1 and melittin. Briefly, bacteria were grown overnight at 37°C and shaken at 200 rpm in TSB. The cultures were then washed twice with The hemolytic activity of synthetic PAJE was assessed against human red blood cells (hRBCs). Jelleine-1 and melittin were used as positive control peptides. Erythrocytes were washed thrice with phosphate-buffered saline (PBS) (10 mM PBS, 150 mM NaCl, pH 7.4), and centrifuged at 1,000 ×g and 4°C for 10 min. A 100-µl cell suspension, diluted with 10 mM PBS (final concentration approximately 8%), was mixed with 100-µl of varying peptide stock solutions in 96-well microtiter plates. The resulting suspension was incubated for 1 h at 37°C. After a 5-min centrifugation at 1,000 ×g and 4°C, release of hemoglobin was monitored by measuring absorbance of the supernatant at 405 nm. The values for 0% and 100% hemolysis were determined using erythrocyte suspensions incubated in 10 mM PBS and 0.1% Triton X-100, respectively.
Recently, a large number of AMPs have been identified and characterized from a variety of insects [5]. Papiliocin was reported to have significant antimicrobial activities against both gram-positive and gram-negative bacterial strains and the fungal strain C. albicans [5,6,8,14,15]. Furthermore, Jelleine-1 was found to be active against most bacterial species (gram-positive and gram-negative) as well as the yeast examined [1].
In this study, we developed a novel hybrid PAJE peptide as an AMP (Fig. 1). PAJE contained the 1-7 amino acids (RWKIFKK) of the N-terminal spiral structure of papiliocin, isolated from the larva of tiger butterfly, and 1-8 amino acids (PFKISIHL) of Jelleine-1 isolated from Royal Jelly, to form a reduced form of positive electrode and hydrophobic amino acid residues. The final amino acid sequence of PAJE was RWKIFKKPFKISIHL-NH 2 , a 15mer peptide, which consisted of hydrophobic (red circle) and hydrophilic (blue circle) amino acids. The net charge and hydrophobicity (%) of the peptide sequence are shown in Table 1. The hydrophobic face facilitates its penetration into the membrane, thereby disturbing the bilayer curvature [10,11,14,16,18]. Higher hydrophobicity has frequently been observed to cause higher antimicrobial activity [14]. The efficacy of AMPs has also been observed to vary with the net positive charge, which enhances its binding to the negatively charged membrane by electrostatic effect [14,15]. PAJE, a synthetic peptide, demonstrated antimicrobial activity against E. coli (gram-negative bacteria) and S. aureus (gram-positive bacteria), as observed from the agar well diffusion assay (Fig. 2). The antimicrobial activity of PAJE was higher than that of Jelleine-1 (160 μg/ml) used as a control. However, PAJE showed antibacterial activity against E. coli and S. aureus even at low concentrations (20-40 μg/ml).
The MIC values are summarized in Table 2. As expected, PAJE proved to be active against all microorganisms tested, with MIC values of 1-4 μM. It evidenced profound growth-inhibitory effects on gram-negative bacteria (MIC value of 1.0 μM). These effects of PAJE were stronger than those of melittin (four times), papiliocin (four times), and Jelleine-1 (eight times). One of the most desired properties of AMPs is its low toxicity to eukaryotic cells. To study the cytotoxicity of the newly designed peptide, we determined its ability to lyse hRBC. PAJE showed no hemolytic activity against hRBC, even at high concentrations ( Table 3), suggesting that it is not detrimental to eukaryotic cells.
Sterilization rate analysis of PAJE against E. coli and S. aureus was performed using a time-kill curve assay (Fig. 3). PAJE showed very high antibacterial activity and time-dependent bactericidal activity; it had a similar microbicidal effect as melittin. Furthermore, we added different concentrations of PAJE to check dosedependence of its activity (Fig. 4). The peptide was found to inhibit E. coli growth by approximately 99.9% after 1 h, at a minimum concentration of 16 μg/ml, and S. aureus growth by approximately 50% after 1 h at a treatment concentration of 16-32 μg/ml (Fig. 4).
In conclusion, we report the development of a novel 15-mer antimicrobial peptide that was highly effective against both gram-negative and gram-positive bacteria, the former being more prone. It exhibited a higher net charge than Jelleine-1, and was as efficient as melittin as a potent antimicrobial peptide. The hydrophobicity (%) of PAJE was higher than that of melittin. Therefore, our synthesized peptide exhibited a lower MIC (1 µM), than melittin, against the four tested microbes. This study suggests that the novel 15-mer short peptide is a highly effective antimicrobial agent. It exhibited no cytotoxicity and its synthesis was relatively inexpensive, due to its small size. The new peptide possesses great potential as an antimicrobial agent.