Studies On The Relationship Between Structure And Function Of Antimicrobial Peptide Cecropin-XJ And Its Antibacterial Mechanism

Cecropin-XJ gene was isolated from Xinjing silkworm and the corresponding full peptide has been expressed in Pichia yeast and E.coli in our laboratraty.Kinetic studies have shown that the Cecropin-XJ has an effective antibacterial activity.It has exhibited an extreme heat-stable property and the ability to kill ampicillin-resistant S.aureus.Moreover,It has been observed that the Cecropin-XJ was highly tolerant to extreme acidic,basic,and high salt environments as well as resistant to 24-hour digestion by artificial gastric juice.With a broad spectrum of antibacterial activities,the Cecropin-XJ is able to inhibit both Gram positive and Gram negative bacteria.Although it has been biologically characterized,the structure-function relationship and antibacterial mechanisms of the Cecropin-XJ have not yet been clarified.It has not known whether Cecropin-XJ antibacterial mode of action is due to their effects on membrane,or their effects on intracellular targets such as DNA or the both.In this study,there are five Cecropin-XJ genes were modified by PCR and three other new genes were synthesized. Growth curves of the transformant E.coli containing different new Cecropin-XJ genes were performed to determine the antibacterial activities.To further characterize the antibacterial activity of modified cecropin-XJ,the bacterial inhibition experiment in vitro was also performed.At the same time,the effects of Cecropin-XJ from Xinjiang silkworm on growth-enhancing,serum hormone levels and physical and chemical index in chicken were performed.Mouse antibodies against Cecropin-XJ were generated by DNA immunization. The interaction of the antimicrobial peptide Cecropin-XJ from Xinjiang silkworm and Staphylococcus aureus DNA was investigated by ultraviolet spectra and fluorescence spectra in vitro.The interaction mode was studied by using ethidium bromide(EB) as an extrinsic fluorescence probe.Ultrastructural observation of S.aureus treated with Cecropin-XJ on Scanning Electron Microscope and Transmission Electron Microscope were performed.The localization of immuno-labelled antimicrobial peptides was studied using Transmission Electron Microscope.The main results are as follows: (1) It was showed that structural parameters includingα-helix,amphipathicity, hydrophobicity,net positive charge,crucial amino acid substitution and so on are interdependent,and therefore,modification of one parameter often leads to significant changes to one or more of the others.Alpha-helix plays an important role in the function of antibacteria,the location of which was of no importance to cecropin-XJ.Amphipathicity may play a more predominant role regarding interaction with target membranes.Hydrophobicity is an essential feature for interactions between antimicrobial peptide and membrane,but its degree must be kept in a certain range.Within the certain range,increasing peptide cationicity is generally associated with increasing antimicrobial potency.However,there is a limit beyond which increasing positive charge no longer confers increased activity.Tryptophan and C-terminal acylation play a crucial role in killing bacteria for Cecropin-XJ.(2) The results showed that the absolute weight of chicken reared by tap water supplemented with 1.5,3.5 mL/L Cecropin-XJ increased significantly than control group(P＜0.05).Compared with the control group,there was no significant difference in serum total protein of experimental groups(P＞0.05),but the concentration of serum urea nitrogen decreased significantly in experimental groups supplemented with 1.5,3.5 mL/L Cecropin-XJ (P＜0.05).Serum IGF-1 levels of experimental groups increased significantly than control group.Experimental groups' serum T3 levels at 28 days were higher than those of control group,but there was no significant difference.Serum T4 levels of experimental groups were significantly lower than those of control group at 28 days(P＜0.05).T3 levels of experimental groups were significantly lower than those of control group chickens at 56 days (P＜0.05),but T4 levels of experimental groups were significantly higher than those of control group chickens.(3) It was showed that the recombinant plasmids were successfully expressed in mouse liver and induced antibody with higher immune reactivity and specificity in mice.The specific antibody levels of mice immunized with the recombinant plasmid were significantly higher than those with empty vector(P＜0.05).Antibody levels of mice immunized with pcDNA3-Cecropin-XJ4R were higher than those of pcDNA3-Cecropin-XJ,but there was no significantly difference.(4) The increase in absorbance of DNA samples at 260nm due to the addition of Cecropin-XJ was measured.It is called hyperchromicity of DNA,which can provide a direct measure of the degree of base-pair unstacking.The unstack results in the loss of duplex helix and then leads to the duplex helix relaxing.At the same time,the interaction mode was studied by using ethidium bromide(EB) as an extrinsic fluorescence probe.With the addition of Cecropin-XJ,the intensity of intrinsic fluorescence absorbance of DNA at 593nm increases greatly.It is suggested that the addition of Cecropin-XJ may stack with the base-pair of DNA or expose the aromatic amino acides,which lead to the enhancing of DNA fluorescence intensity.Subsequently the competition between Cecropin-XJ and EB to combine with DNA was found.It suggested that the style was groove binding and intercalation in the interaction between Cecropin-XJ and double helix DNA.Furthermore,in this study,the binding constant and binding number of Cecropin-XJ complex with DNA was determined.The binding constant and number of EB complex with DNA changed with addition of Cecropin-XJ.It showed that the interaction between Cecropin-XJ and DNA was based on intercalation and non-intercalation.Taken together,it indicated that Cecropin-XJ can bond to S.aureus chromosome DNA,which suggests existing DNA synthesis inhibition mechanism. Meanwhile,these results contribute to explain the molecular mechanism of antimicrobial peptide from the interaction style and structural charateristic of Cecropin-XJ and S.aureus DNA.(5) It was showed that the ability of cecropin-XJ to kill S.aureus is effective and time-dependent.It may be the "barrel-stave pore" mechanism that cecropin-XJ quickly penetrated and damaged the membrane.And then it was observed that cecropin-XJ make effects on the cytoplasmic membrane,disturbing the ordered arrangement of membrane lipid and changing the permeability of cytoplasmic membrane with the cytoplasmic contents of the cells leaking out.Finally,the affected S.aureus was died due to leakage of the cell contents. The intact cell wall was left,not losing their shape after treated with cecropin-XJ,which strongly showed that no cell wall disintegrated,althought cecropin-XJ acted on S.aureus and led to cell death.These results indicated cytoplasmic membrane is the main target attacked by cecropin-XJ.(6) The immunolabelled localization of antimicrobial peptides was studied using transmission electron microscopy.Staphylococcus aureus was exposed to Cecropin-XJ. Cecropin-XJ was found in the cytoplasm and membrane of bacteria.Cecropin-XJ could effectively kill Staphylococcus aureus in the dosage-and time-dependent way.Based on these results,modification of Ceeropin-XJ to improve antimicrobial activity may be important in the rational design of novel therapeutic agents.To our knowledge,this study represents the first report on two different mechanism of action of Cecropin-XJ to kill S. aureus.First,membrane mechanism of Cecropin-XJ was emphasized by the SEM and immuno-TEM.Second,Cecropin-XJ may interact with DNA to inhibit the DNA replication and transcription,and then inhibit S.aureus growth and development.In addition to increasing our understanding of a novel mechanism of action of Cecropin-XJ,the study presents a potential antibiotic to overcome S.aureus,especially in killing antibiotic-resistant S.aureus, and contributes to develop a novel antibiotic for future therapeutic purposes.These findings could lead to a broad application prospect in the medical industry and domestic animal industry of Xinjiang.