Anti-pathogenic Microorganism Activity And Mechanism Displayed By α-helical Antimicrobial Peptides And Human α-defensin 4

Antimicrobial peptides(AMPs) had been extensively studied as broad-spectrum anti-bacterial and anti-viral agents and rarely caused the emergence of drug-resistant microorganism. Numerous studies about its application in the fields of medicine, agriculture and materials etc had been achieved. Nowadays the pig industry in China faced different kinds of challeges including the emerging of drug-resistant bacterial strains and outbreak of epidemic viral diseases. Therefore, novel agents need to be developed. Cecropin B, moricin, piscidin, caerin, maculatin, lactoferricin B and indolicidin were used in this study to test their antibacterial and antiviral activities against swine-origin pathogens. And HNP4 was chosen as the representative peptide to study the structure-activity relationships of AMPs. Three parts of work related to the contents mentioned above were conducted: 1. Antimicrobial activity and mechanism displayed by cecropin B and moricin against swine-origin bacteriaAnti-bacteria activities of the AMPs were tested. Eight species of bacteria including H. parasuis, A. pleuropneumoniae and S. suis etc., were used in this study. According to the results, moricin displayed activity against all the organisms tested. Cecropin B got a better inhibition activity against the gram-negative bacteria. Through the kill-curve study, they were found to kill all the bacteria within a short period.Relationship between the biochemical characteristics of the strains and its sensitivity to cecropin B. Antimicrobial activity of cecropin B was tested against standard serotypes and clinical isolates of H. parasuis. Cecropin B showed similar activity against all the isolates with the MICs being 2 μg/ml-16 μg/ml. At the same time, hexadecane adsorption assay and electrophoresis were conducted to study the hydrophobicity and electricity of the different strains. A correlation was established between these physical-chemical parameters of the isolates and its sensitivity to cecropin B. It was found that if the isolate was more hydrophobic and/or more negatively charged, it would be more sensitive to cecropin B.Morphogical changes of HPS caused by cecropin B. TEM, SEM and AFM were used to investigate the antibacterial mechanism adopted by cecropin B. It was observed that cecropin B could damage bacterial membrane, causing leakage of cell contents and "ghost cell”. Some internal changes like electron dots could also be observed, which indicated that besides the membrane cecropin B could also interact with the inner targets. These results showed that cecropin B could be developed as a promising drug against gram-negative bacteria. 2. Antiviral activity and mechanism displayed by the AMPs against swine-origin virusesThe antiviral activities of the AMPs were tested. As moricin and cecropin B did not show any antiviral activity, another five AMPs(piscidin, maculatin, caerin, lactoferricin B and indolicidin) of different origin were selected to test their inhibition activities against swine-origin viruses, i.e. pseudorabies virus(PRV), porcine epidemic diarrhea virus(PEDV), porcine reproductive and respiratory syndrome virus(PRRSV) etc. Through TCID50 assay, it was found that piscidin, maculatin, caerin could inhibit most of the viruses tested. Among all the viruses tested, piscidin and caerin showed the best inhibition activity against PRV with the surviving rate being 2% and this inhibiton activity was not strain-specific.The PRV repliction stages intervened by AMPs. Three different stages were tested: cells were treated by AMPs before PRV infection, cells were treated by AMPs after PRV infection and virus particles were directly treated by AMPs. According to the results, maculatin, caerin and piscidin directly interacted with the viruses. By nonlinear regression analysis, the activity of the three AMPs against PRV in descending order was as follows: piscidin> maculatin>caerin. And it was found that PRV induced apoptosis would also be inhibited by the three AMPs.The protective effect of piscidin was tested in vivo. The results showed that piscidin at 2.5 μg/ml could protect 90% of the mice and at concentrations above 5 μg/ml it would fully protect mice from RPV-induced death. All these results indicated that piscidin could be developed as an antiviral drug. 3. Study about structure-function relationship of HNP4Antibacterial activity of HNP4 was affected by amino acids and structure. All the HNP4 ala-scanning mutants were synthesized and purified. Then, antibacterial assay against S. aureus and E. coli were performed. It was observed that HNP4 had better activity against gram-positive bacteria over gram-negative bacteria. Also it was found that arginine was important for the antibacterial activity of HNP4. All the arginine mutants showed much lower activity against E. coli or S. aureus than that of the wild type. The results proved that cationic amino acids were important for the antibacterial activity of HNP4. And F26A-HNP4 lost almost all the activity against S. aureus. However, the inhibition activity of F26A-HNP4 against E. coli was not affected. MeLeu20-HNP4, Leu20 of which was methylated to avoid dimerization, was synthesized. Antibacterial assay revealed that dimerization of HNP4 did not affect its inhibition activity against gram-negative bacteria while the S. aureus inhibition activity of MeLeu20-HNP4 decreased about three times. The results indicated that possibly HNP4 inhibited E. coli and S. aureus by two different mechnisms.Activities of LF inhibition and gp120 binding of HNP4 were affected by amino acids and structure. By non-linear regression analysis, it could be found that the results were consistent with the result of the antibacterial assay as the activity of F26A-HNP4 was the worst. The performance of arginine mutants was a little better than that of F26A-HNP4. And dimerization would also affect the LF inhibition and gp120 binding activities of HNP4. All these results indicated that Phe26 was most import amino acid in HNP4 sequence, followed by the cationic Args. Dimerization would also affect some funcions of HNP4.In conclusion, cecropin B and piscidin were found to have obvious inhibition activity against HPS and PRV. Through studies about the structure-function relationship of HNP4, Phe and Arg were identified to be the important factors affecting the functions of HNP4. Strategies about how to study the structure-function relationship and optimize the sequences of AMPs had been built. It paved the way for the further optimization and application of AMPs in porcine industry.