| Because of the extensive use of traditional antibiotics, the frequent emergence ofresistant bacteria has resulted in a significant threat to breeding industry and humanpublic health. Therefore, there is a vital need for new antimicrobial agents witheffectivity to conquer drug-resistant bacteria and novel modes of action so as toreplace the traditional antibiotics. During the development of new antimicrobialagents, antimicrobial peptides (AMPs) has been received more attention for its broadspectrum bactericidal activities. AMPs are a class of defense peptides that defenseagainst the invading microorganism and a component of innate immune system.Compared to traditional antibiotics, AMPs have some good qualities, such as smallmolecular weight, good water-solubility, thermal stability and unique antibioticsmechanism. They also possess broad-spectrum of antimicrobial activities to clinicalresistance bacteria. As the development of the research on AMPs, AMPs also foundrestrain virus, fugus, tumor and regulating action to the immunity. Thus，AMPs havebecome a promising alternatives to conventional antibiotics.The objective of this study is to enhance the antimicrobial activities of P3, anantimicrobial peptide isolated from bovine hemoglobin α-subunit, by moleculardesign and evaluating its hemolysis, cytotoxicity, and selectively inhibition to Helacells. The structural features of P3were studies by CD spectra. The membraneperturbation action model was explored elementary by the test of membranepermeability, observation of SEM, and monitoring of transmembrane potential. Theintracellular action was measured by the disturbance of P3to the balance ofcytoplasmic Ca2+. At last, the mechanism of bactericidal action of P3could beelucidate combined the action to membrane and in cytoplasm. The theraputics effectto ICR mice of P3and its analogs were evaluated in an E. coli clinical strain infectedmouse model.The four derivatives, JH-0, JH-1, JH-2, JH-3, synthesized by optimizing thestructure of P3showed stronger antimicrobial activities than P3to test bacterialstrains with low hemolysis and cytotoxicity. Besides, JH-3showed excellentselectively inhibition to Hela cells. The analysis of membrane action mechanismfound that P3formed a typical α-helix induced by phospholipids in the membrane-mimicking environment. P3acted on bacteria, increased the membranepermeability, and formed transmembrane pore or regional lysis in bacterial membrane,which caused physical damage to the integrity of membrane. P3also induced thedepolarization to transmembrane potential. It induced intracellular calcium ionsoscillation and calcium overload. Ultimately, P3caused the death of bacteria bydiversified action mode on-memrbane and off-membrane.An ICR mouse model of infection was employed to evaluate the antibacterialactitivty of P3and its analogs in vivo. The P3and its analogs showed more weakcytotoxicity in vivo than PMB, a clinical drug, and the mouse had higher absorbilityof peptidein intraperitoneal injection than in subcutaneous injection. P3and itsanalogs saved no less than60%of infected mouse, especially, JH-3saved90%, andall mouse died during20h in the group of Amp and control. After injected2h, P3andits analogs decreased the bacterial content in mouse’s blood distinctly, JH-3did verysignificant. The results indicated that P3and its analogs showed effective therapeuticsto conquer infection caused by drug-resistant bacteria in vivo. |
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