In Vitro Antibacterial Study Of Antibacterial Peptide Magainin Ⅱ Modified By Cell Membrane Penetrating Peptide TAT Against Brucella S2

Brucellosis is a chronic and infectious zoonotic disease caused by the intracellular parasite Brucella spp.Brucellosis has been classified as a second-class animal disease in China.Animals infected with brucellosis must be destroyed harmlessly to avoid spread.At present,it is mainly prevented by immunization in animal herds.Humans are infected with Brucella mainly through the direct contact with infected animals and its products.When people get infected,symptomatic treatment combining with antibiotic therapy are usually applied.However,the treatment cycle is too long and the prognosis is very poor,often get relapse.Therefore,it is an urge to develop new drugs that can effectively remove intracellular Brucella.Magainins are a class of antimicrobial peptides from the African frog Xenopus laevis,which have significant inhibitory effects on a variety of microorganisms,including bacteria,fungi,viruses and protozoa,and also have certain antitumor activity.TAT is a widely-studied and-used transmembrane peptide.It not only has certain antibacterial and antitumor activities,but also can combine with the corresponding therapeutic drugs and transport them from extracellular to intracellular without affecting the function of the drug itself.In this study,the transmembrane peptide TAT was hybridized with the antimicrobial peptide Magainin II(MAG2),named TATMAG2.The inhibitory effect of TAT-MAG2 on Brucella S2 was evaluated in vitro,aiming to have a preliminary exploration of a new and effective drug for the treatment of brucellosis.In order to get a high purity of peptides,we chose to chemically synthesize peptides TAT,MAG,and TAT-MAG2,with a purity of more than 95%.The sequence of TAT-MAG2 contained the MAG2 sequence with TAT sequence added into the N-terminal.The results of the minimal inhibitory concentration test showed that three peptides had no significant inhibitory effect on Staphylococcus aureus of Gram-positive bacteria.For Gram-positive bacteria,TAT had no significant inhibitory on E.coil and Brucella S2(MIC>100 μM).MAG2 showed similar inhibitory effect on E.coil and Brucella S2,with the same MIC of 50 μM.TAT-MAG2 also represented a remarkable inhibition of E.coil and Brucella S2,with MICs of 12.5 μM and 6.25μM,respectively.The MICs of TAT-MAG2 for E.coil and S2 were separately 1/4 and 1/8 times to those of MAG2.Three peptides with the concentration of 4 × MIC were acted on S2 to test the killing effect.The kill kinetics curve showed that TAT had no significant inhibitory effect on S2.MAG2 showed inhibitory effect after 1 h,6 h and 9 h co-incubation.The growth of S2 was significantly inhibited when co-incubated with TAT-MAG2 within 15 h,compared to the control group.Fluorescence microscope observation showed that the three peptides could co-located with S2.In order to further determine the damage effect of TAT-MAG2 on S2,we carried out the scanning electron microscopy analysis,and found that TAT-MAG2 could alter or even rupture the S2 cell membrane structure.These results above suggested that the antibacterial ability of the hybrid TAT-MAG2 on intracellular bacteria S2 was enhanced compare to individual action.The hemolytic activity and cytotoxicity of the three peptides were also analyzed.the hemolytic activities of the three peptides on mammalian erythrocytes were all less than 5%within the concentration of 100 μM.Under the MIC concentration of each peptide,no cytotoxicity was found on RAW264.7 cells.Brucella is the intracellular bacteria,which mainly enters into macrophage to replicate and proliferate after infection.Therefore,an effective bactericidal drug must be able to enter the cell to kill the intracellular bacteria at the same time.To determine the inhibitory effect of TATMAG2 on intracellular S2 proliferation,the RAW264.7 macrophage was selected as the infection target cell of Brucella S2,and the three peptides were then used to act on the infected macrophages,respectively.The bacterial separating result showed that the number of live S2 in infected RAW264.7 cells was significantly lower than that in the control group at the time point of 2,4 and 8 hours.The results of q PCR also verified the bacterial separating results,indicating that TAT-MAG2 had an inhibitory effect on S2 intracellular infection.Based on the above experimental results,compared with TAT and MAG2,the hybrid peptide TAT-MAG2 has a good bactericidal effect on the intracellular bacterium Brucella S2 in vitro,and exhibits a low hemolytic activity and a low cytotoxicity within the working concentration,making the TAT-MAG2 become a potential drug alternative for clinical treatment of Brucella infection.