| The accelerated emergence of pathogenic bacteria caused by the extensive use of traditional antibiotics is a major threat today. Therefore, the development of a new class of broad-spectrum antibiotics is an urgent need. Antimicrobial peptides(AMPs)are an important component of the natural defenses of most living organisms,it plays an important role in against invading pathogens. Compared to antibiotic, AMPs have the advantage of broad-spectrum antimicrobial activity, lower molecular weight, low toxicity, good thermal stability and not have the drug resistance.Previous reports have shown that the membrane-proximal external region(MPER,Ac-665 WASLWNWFNITNW LWYIK683-NH2) of HIV-1 has the membrane-disrupting properties. Indolicidin, is a naturally occurring AMP isolated from bovine neutrophils.Indolicidin is an analog of MPER and is also capable of disrupting membranes and has an unusually high number of Trps as well. Based on the membrane-disrupting properties of the MPER, we hypothesized that synthetic peptides derived from this region, may have functional properties similar to those of known AMPs.Considering the molecular weight, the 4E10 epitope and the hemolytic of AMPs,we choose the the 4E10 epitope(NWFNITNWLWYIK) for the study of AMPs.Although the sequences of AMPs vary greatly, certain amino acids, such as lysine(Lys) or arginine(Arg), are key components of AMPs. In particular, Lys-rich AMPs exhibit rapid, nonhemolytic, broad-spectrum microbicidal properties. Thus, we aimed to increase this activity by adding three Lys to the C-terminus of the 4E10 epitope to generate an AMP with 16 amino acids(AP16: Ac-NWFNITNWLWYIKKKK-NH2).In this study, we measured the minimal bactericidal concentration(MBC) of AP16 against E.coli DH5α, B.subtilis ATCC 6633 and S.epidermidis ATCC12228.The results indicated that the MBC to S.epidermidis was 256 μg/mL(115.07 μM), and AP16 showed better antimicrobial activity to B.subtilis, the MBC was 32 μg/m L(14.38 μM), but AP16 had no antimicrobial activity to E.coli.Based on the result of antimicrobial activity of AP16, we changed the sequence of AP16 or added the cationic amino acid to enhance the antimicrobial activity of AP16.Compared to AP16, the MBC of AP16-A is 16 μg/m L(7.34 μM) to E.coli, the MBC increased by 8 times [4 μg/mL(1.834 μM)] and 32 times [8 μg/m L(3.67 μM)] to B.subtilis and S.epidermidis, respectively.The results of cytotoxicity in vivo and in vitro showed that AP16-A had low toxicity to mammalian cell and no hemolytic activity. And in the detection of stabiliy,AP16-A was stable at 37℃ after 15 days, the MBC of AP16-A was not changed.Based on the results of NPN uptake assay and the assay of in vitro binding between AP16-A and LPS, we proposed the antimicrobial mechanisms, AP16-A could bind to LPS and permeabilize the outer membrane of gram negative bacteria. Furthermore,results of dose-dependent bacterial killing, confocal fluorescence microscopy, the SYTOX Green uptake assay and Gel retardation assay indicated that AP16-A killed Gram-negative bacteria by the combined effects of relatively slow membrane permeabilization and interaction with an intracellular target, while it killed Gram-positive bacteria by a fast membrane permeabilization process, which achieved relatively more rapid bacterial killing kinetics.In conclusion, we synthesised an AMP AP16-A which had good antimicrobial activity, and we proposed the potential antimicrobial mechanisms of AP16-A. The results of this study support the potential use of AP16-A as an AMP. |
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