BackgroundAntimicrobial peptides (AMPs) are considered promising candidates for the development of novel anti-infective agents. In arthropods such as ticks, AMPs form the first line of defense against pathogens in the innate immune response. Persulcatusin (IP) was found in the Ixodes persulcatus midgut, and its amino acid sequence was reported. However, the complete structure of IP has not been identified. We evaluated the relation between structural features and antimicrobial activity of IP, and its potential as a new anti-methicillin-resistant Staphylococcus aureus (MRSA) agent.MethodsThe structure of IP was predicted using homology modeling and molecular dynamics. IP and other tick AMPs were synthesized using a solid-phase method and purified by high-performance liquid chromatography. Methicillin-susceptible S. aureus (MSSA) and MRSA were used for the minimum inhibitory concentration (MIC) test and short-time killing assay of IP and other tick peptides. The influence of IP on mammalian fibroblasts and colon epithelial cells and each cell DNA and its hemolytic activity towards human erythrocytes were also examined.ResultsIn the predicted IP structure, the structure with an S-S bond was more stable than that without an S-S bond. The MIC after 24 h of incubation with IP was 0.156–1.25 μg/mL for MSSA and 0.625–2.5 μg/mL for MRSA. Compared with the mammalian antimicrobial peptide and other tick peptides, IP was highly effective against MRSA. Moreover, IP showed a dose-dependent bactericidal effect on both MSSA and MRSA after 1 h of incubation. IP had no observable effect on mammalian cell growth or morphology, on each cell DNA and on human erythrocytes.ConclusionsWe predicted the three-dimensional structure of IP and found that the structural integrity was maintained by three S-S bonds, which were energetically important for the stability and for forming α helix and β sheet. IP has cationic and amphipathic properties, which might be related to its antimicrobial activity. Furthermore, the antimicrobial activity of IP against MRSA was stronger than that of other antimicrobial peptides without apparent damage to mammalian and human cells, demonstrating its possible application as a new anti-MRSA medicine.

Persulcatusin (IP), which is an antimicrobial peptide found in Ixodes persulcatus midgut, is active against Gram-positive bacteria such as Staphylococcus aureus. Multidrug-resistant bacteria in particular methicillin-resistant S. aureus (MRSA), vancomycinintermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) are a worldwide clinical concern. In the present study, to explore the potential of IP as a new agent against multidrug-resistant S. aureus infections, we evaluated the antimicrobial activity of IP against multidrug-resistant S. aureus strains by MIC 90 , morphological observation with scanning electron microscope (SEM), and the calcein leakage assay of membrane integrity. Among the six antimicrobial peptides used in this study, IP exhibited the lowest MIC 90 values for both vancomycin-susceptible and -resistant S. aureus strains. The IP MIC 90 against a VISA strain was equivalent to vancomycin, while the MIC 90 against VRSA was relatively low. SEM observations indicated that bacterial cells exposed to IP were crumpled and showed prominent structural changes. Moreover, IP influenced the cell membranes of both MRSA and VRSA in a mere 5 min, leading to leakage of the preloaded calcein. Although a VISA strain was resistant to the action of IP on cell membrane, the MIC 90 of IP was lower than that of Nisin, suggesting that IP had another bactericidal mechanism in addition to cell membrane attack. Our results indicate that the synthetic tick antimicrobial peptide, IP exhibits strong antibacterial activity against multidrug-resistant S. aureus strains, including VRSA, via both cell membrane attack and another unknown mechanism. IP represents a promising candidate for a new anti-VRSA therapy. The appearance of antibiotic-resistant bacteria has resulted in bacterial diseases re-emerging as a threat after the many decades since the introduction of the first antibiotic penicillin. 1 The development of new antibacterial agents is urgently required, and antimicrobial peptides are viewed as ideal candidate agents. 5 Antimicrobial peptides are integral components of the innate immune system of all living organisms, including mammals, plants and insects. 6 Antimicrobial peptides result in strong natural defense in arthropod, particularly antimicrobial peptides of silk moth 7,8 and beetle, 9 which belong to the defensin family. Persulcatusin (IP), a tick antimicrobial peptide found in the midgut of Ixodes persulcatus, exhibits antimicrobial activity against Gram-positive bacteria such as S. aureus. 10-12 Furthermore, we have reported previously that S. aureus strains could not be isolated from I. persulcatus during feeding. 13 This is attributable to the antimicrobial activity of IP, which is highly expressed during blood feeding. 10,11 We hypothesized that IP could exhibit antimicrobial activity against VISA and VRSA, as well as methicillin-susceptible S. aureus and MRSA.In this study, we evaluated the antimicrobial activity of IP against multidrug-resistant S. aureus strains using MIC, scanning electron m...