Activity And Mechanism Of Scorpion Peptide Bmkn2and Its Derivative Peptide Kn2-7Against Antibiotic Resistant Bacteria

Bacteria infection has been posing a threat to human health. New drug-resistant pathogens have continued to emerge with the abuse of antibiotics. The rates of Methicillin-resistant Staphylococcus aureus (MRSA) infections increased rapidly during the past decade and there is little antibiotic treated with. Drug-resitant pathogens have become an important public health problem and new types of antimicrobial agents are urgently needed to respond to the threat of pathogens that evolve resistance against conventional antibiotics. Antimicrobial peptides (AMPs) are ubiquitous among a wide range of species, including insects, plants, humans, and even single-celled organisms. AMPs play an important role in host defense against pathogens such as bacteria, fungi, viruses and parasites. Compared with conventional antibiotics, some AMPs could kill antibiotic-resistant pathogens and are difficult to induce resitance. These characters and the large size of the AMP family make AMPs attractive to be developed as new types of antibiotic agents.The scorpion Buthus martensii Karsch has been used as a Chinese traditional medicine for thousand years. The toxin peptides secreted by its venom gland are the main active ingredients. Some of the toxin bioactive peptides without disulfide bridges from the venom of the scorpion are typically9to30ami no acid residues in length and these peptides have bactericidal activities. Therefore, it is significant to screen natural antimicrobial peptides from scorpion venom and get derived peptides with higher activity and lower hemolytic activity through molecular design.A precursor peptide composed of70ami no acid resedues, BmKn2, was charcterized from the venom cDNA library of the scorpion Buthus martensii Karsch. BmKn2consist three parts:the signal peptide with23amino acid residues, the without disulfide bridges mature peptide with13residues and the propeptide with34residues. The venome of the scorpion Buthus martensii Karsch was collected through electrical stimulation. Then BmKn2was characterized from the venome through HPLC combined with mass spectrometry. The in vitro antimicrobial activity of synthetic BmKn2was determined. We showed that BmKn2exhibited bactericidal activity specificly against Gram-positive bacteria and BmKn2also showed high antimicrobial activity against clinical antibiotic-resistant strains including MRSA. To determine the in vivo antimicrobial activity of BmKn2, an MRSA mouse infection model was established. Seven days after treated with BmKn2, the the group of mice were found totally survived. The results indicated that BmKn2could inhibit the growth of MRSA effectively in vivo. Additionally, the bactericidal mechanism of BmKn2was studied. The results of killing assay and emzyme release assay sggueseed that BmKn2could disrupt the bacteria to release the contents and kill bacteria rapidly. Furthemore, semi-thin sections of BmKn2-treated bacteria showed a release of the cell content upon membrane structure disruption.To improve the activity, broaden the antibacterial spectrum and decrease the hemolytic activity of BmKn2, molecular design was carried out based on the skeleton of BmKn2. Kn2-7was designed with the aim of enhancing the net positive charge of the hydrophilic side of the peptide while maintaining the amphipathic character of the peptide. The amino residues Gly3, Ala4and Ser10of BmKn2were substituted by Lys3, Arg4and Arg10. The antimicrobial functional screening results for BmKn2and its derivatives showed that the Kn2-7mutant exhibited the strongest inhibitory activity against both S. aureus and E. coli. The hemolytic activities of Kn2-7and BmKn2were tested against human red blood cells, we found Kn2-7had little hemolytic activity at high concentration and the HC50value of Kn2-7was5times over BmKn2. To determine the in vivo antimicrobial activity of Kn2-7, an S. aureus mouse skin infection model was established. After4days continuous treatment, the skin wound of the mice treated with Kn2-7healled, the histological structure restored integrity and the number of living colony dropped to normal level. All of these results showed that the devirative peptide Kn2-7exhibits favorable antimicrobial activity in vtro and in vivo and has the therapeutic potential to treat skin infection caused by S. aureus as a topical antibacterial drug.Compared with wild type BmKn2, Kn2-7not only showed increased inhibitory activity against Gram-positive bacteria, but also inhibited the growth of Gram-negative bacteria In this study, the results of secondary structure analysis, bactericidal assays, enzyme release assays provided solid evidence that the inhibitory effect of Kn2-7on the bacteria was mediated through rapid killing. However, the binding mode of Kn2-7to the surface of Gram-positive bacteria seems different from that of Gram-negative bacteria The results of biolayer interferometry and competition binding assay proved to be so. The findings of TEM indicated that the bacterial cell wall of S. aureus was disrupted immediately upon treatment with Kn2-7. Kn2-7accumulated on the surface of E. coli, combined with the outer membrane, and then formed microspheres surrounded the bacteria.We speculate the bactericidal mode isthat Kn2-7bind to LPS of the cell wall firstly, and then they form complexes. Finally, the complexes fuse with the membrane and form the structures of microspheres with the fragment of the cell membrane. This mode is consistent with the model of carpet mechanism in general. But to the best of our knowledge, the structures of the microspheres were first observed in the antimicrobial mechanism study and our results provide conclusive evidence for the model of carpet mechanism.In conclusion, we described both in vitro and in vivo antimicrobial activities of BmKn2, a scorpion venom peptide. Kn2-7was design based on the molecular skeleton of BmKn2to improve the antibacterial activity and deaease the hemolytic activity. Kn2-7could inhibit the growth of both Gram-positive bacteria and Gram-negative bacteria including antibiotic resistant clinical isolates MRSA. The findings of TEM indicated the binding mode of Kn2-7to the surface of E. coli was different from that of S. aureus. The peptide Kn2-7disrupted the cell surface structure of S. aureus and E. coli via binding to LTA and LPS, respectively. Taken together, these data strongly suggest that Kn2-7peptide can be developed as a topical therapeutic agent for treating bacterial infections. And the mechanism study of Kn2-7provides conclusive evidence for the model of bactericidal mechanism.