The Effect Of The Introduction Of Histidine On The Biological Activity,Structure-activity Relationship Of Antimicrobial Peptides And The Interaction Between Peptides And Metal Ions

Nowadays,drug-resistant bacteria have become a serious clinical problem that seriously threatens people's health and life.Antimicrobial peptides?AMPs?are considered as potential substitutes for conventional antibiotics against the threat of drug-resistant bacteria.Most cationic antimicrobial peptides in nature do not contain histidine,and only part of them contains histidine.The side chain imidazole group of histidine often makes these antimicrobial peptides have different properties from those containing other cationic amino acids,such as being susceptible to the influence of metal ions.However,the effect of histidine on the structure and function of cationic antimicrobial peptides is not yet clear.Therefore,this study intended to take two natural cationic antimicrobial peptides Polybia-MPI?MPI?and Protonectin?Pro?as the research object.In addition,this research explored the effect of introducing histidine into natural and histidine-free cationic antimicrobial peptides on the activity,structure-activity relationship,stability,toxicity of antimicrobial peptides and the response of peptides to metal ions.MPI and Pro are cationic antimicrobial peptides isolated from the venoms of the social wasps Polybia paulista and Agelaia pallipes pallipes,which have good antibacterial,antifungal activity and low cytotoxicity.The sequence of MPI contains three lysines and the sequence of Pro contains a lysine.First,we discussed the effect of replacing lysine with histidine on MPI's antimicrobial activity,stability,secondary structure,cytotoxicity,and cell selectivity.It was found that,compared with MPI,His-Polybia-MPI?HMPI?only exhibited antifungal activity,but no activity against Gram-negative and Gram-positive bacteria.Compared with MPI,HMPI was more stable to trypsin.The cytotoxicity of HMPI on mouse fibroblast NIH 3T3 was significantly reduced.The results of the secondary structure study showed that the antimicrobial peptides exhibited a typical?-helical structure in the membrane-simulated environment after histidine replacement,but the content of?-helix decreased compared to MPI.Examination of the binding force between MPI and HMPI and LPS revealed that the binding force between HMPI and LPS was much lower than that between MPI and LPS.And the dissociation constants?Kd?of their binding to LPS were 3.22?M and 21.5?M,which may partially explained that the antibacterial activity of HMPI against bacteria was greatly reduced.In addition,it was also found that the activity of HMPI to induce reactive oxygen species in fungal cells was equivalent to MPI,which might be one of the reasons why it could maintain considerable antifungal activity with MPI.It was worth noting that the introduction of histidine reduced the isoelectric point of HMPI,but made HMPI not show cationicity in a neutral partial alkali environment,which might be the main reason for its decreased or lost antibacterial activity in a neutral environment.Then,we explored whether HMPI could interact with metal ions and how metal ions affected the biological activity of HMPI.In vitro antifungal activity studies showed that Cu²⁺,Zn²⁺,Al³⁺,Mn²⁺,Cr³⁺,Ag⁺,Cd²⁺and Pb²⁺had no effect on the antifungal activity of HMPI.But what was interesting was that the OD value detection enzyme stability experiment,colony counting experiment and RP-HPLC analysis showed that copper ion could significantly improve the stability of HMPI to trypsin.The reason for the increased stability of HMPI-Cu²⁺complex enzymatic hydrolysis might be due to the combination of HMPI and Cu²⁺affecting the interaction between trypsin and HMPI.The CD experiment results showed that in the absence of Cu²⁺,HMPI had a disordered conformation in water,while in the presence of CuCl₂ and CuSO₄,the CD spectrum of HMPI had negative peaks at 208 and 222 nm,showing a?-helical conformation.Furthermore,we studied the binding of Cu²⁺and HMPI through ESI-MS,¹H NMR and ITC experiments,and confirmed that Cu²⁺could bind to HMPI.Hemolysis experiment showed that Cu²⁺could reduce the hemolytic toxicity of HMPI.Cytotoxicity experiment revealed that Cu²⁺not only did not affect the cytotoxicity of HMPI on mammalian cells,but even reduced the cytotoxicity of HMPI.In addition,active oxygen experiments showed that Cu²⁺could promote HMPI to produce ROS in Candida albicans cells,which might help to improve its antifungal activity.Our research also showed that the effect of metal ions on cationic antimicrobial peptides with different amounts of histidine in the sequence were different.Compared with HMPI,although the antimicrobial peptide HPro containing only one histidine was not affected by Cu²⁺,Zn²⁺affected its activity.In vitro antifungal experiments showed that Zn²⁺could inhibit the antifungal activity of HPro,and its activity against Candida glabrata decreased by more than 8 times,while Cu²⁺,Ag⁺,Cd²⁺,Pb²⁺,Mg²⁺,Ca²⁺,Ni²⁺had no effect on its antifungal activity.PI uptake experiments showed that HPro could not destroy the integrity of fungal cell membranes in the presence of Zn²⁺.Similarly,the in vivo antifungal experiments showed that Zn²⁺also had a significant inhibitory effect on the antifungal activity of HPro in mice.The hemolysis experiment showed that Zn²⁺significantly increased the hemolytic toxicity of HPro.The active oxygen experiment showed that Zn²⁺could significantly reduce the amount of HPro-induced fungi to produce active oxygen,which might be related to the reduction of HPro+Zn²⁺antifungal activity.The fungal representative polysaccharide binding experiment showed that with the increase of?-1,3 glucan concentration,the antifungal activity of HPro gradually decreased,while the antifungal activity of HPro+Zn²⁺gradually recovered.However,¹H NMR spectroscopy could not confirm the binding of Zn²⁺to HPro.How Zn²⁺affected the activity of HPro remained to be further studied.In summary,in this study,the antimicrobial peptide was modified by histidine replacement,which reduced the isoelectric point of the antimicrobial peptide HMPI.It did not exhibit cationic characteristics in a neutral or alkaline environment,and the antibacterial activity of HMPI could be adjusted by changing the pH value.At the same time,histidine could also reduce the cytotoxicity of the antimicrobial peptide HMPI and increase its enzymatic stability.On this basis,our study also showed that after the introduction of histidine,in the tested metal ions,although the antifungal activity of the antimicrobial peptide HMPI was not affected by Cu²⁺,the combination of Cu²⁺could increase its stability to trypsin,and reduce its hemolytic toxicity.However,unlike HMPI,the cationic antimicrobial peptide HPro containing a single histidine was not affected by Cu²⁺,but was affected by Zn²⁺,and this effect was also different from the effect of Cu²⁺on HMPI.Zn²⁺could reduce the antifungal activity of HPro and increase its hemolytic toxicity.Therefore,it could be inferred that the introduction of histidine has different effects on the antimicrobial activity and toxicity of antimicrobial peptides with different sequences and the response of peptides to metal ions.Although the effect of histidine on cationic antimicrobial peptides needs further study,this study could provide a certain research basis for exploring the important role of histidine in peptide modification.