Study On The Tnteraction Between NgAgo Protein And IntI-1 Protein And The Inhibitory Effect Of NgAgo Protein On The Binding Of IntI-1 Protein To Target DNA Sequence

Animal bacterial infectious diseases restrict the development of animal husbandry,and at the same time bring potential threats to human health.The emergence of antibiotics,to a certain extent,saved the animals harmed by pathogenic bacteria and brought benefits to human beings.However,due to the unreasonable use of antibiotics,pathogenic bacteria gradually developed antibiotic resistance(AMR),in which bacterial multidrug resistance(MDR)maked the treatment of some bacterial diseases into the dilemma of no drug available.Multidrug-resistant pathogens acquired resistance mainly through horizontal transfer of resistance genes mediated by class I integron,in which the function of class I integron was mainly mediated by the integrase IntI-1 protein.NgAgo protein was known to be an endonuclease,which consisted of four domains,namely N terminal,PAZ,MID and PIWI,and the active center of the enzyme exists in the PIWI domain.Previous studies in our laboratory had shown that NgAgo can enhance the ability of RecA to mediate the exchange of homologous DNA strands through the interaction of the PIWI domain with RecA,and improved Homologous recombination efficiency,and its function didn’t depend on the activity of NgAgo nuclease.When analyzed the mass spectrometry data of the NgAgo protein interacting molecules,it was found that the integrase IntI-1 protein is the interacting molecule of the NgAgo protein.It is similar to the study of the interaction and effects of the NgAgo protein and the RecA protein,suggesting that NgAgo protein may affect the efficiency of DNA recombination mediated by Intl-l protein by interacting with Intl-l protein,and then play a certain effect in the process of integron-mediated bacterial resistance.Based on this,this thesis mainly studied the interaction between NgAgo protein and Intl-l protein and its inhibitory effect on binding to target DNA sequence,laying a foundation for the analysis of the mechanism of bacterial resistance and the role of NgAgo protein in bacterial resistance,the research contents were as follows:1)In order to verify the interaction between NgAgo protein and Intl-l protein,this study first constructed a NgAgo protein expression plasmid fused with FLAG tag——pET28a-NgAgo-FLAG,and synthesized a IntI-1 protein expression plasmid fused with His tag——PBAD-IntI-1-His,and then co-transformed two plasmids into BL21 strain,and induced the co-expression of NgAgo protein and IntI-1 protein.This study Used FLAG antibody resin and NTA resin to conduct positive and negative fishing experiments on the supernatant of the co-expression strain,verified the interaction between NgAgo protein and IntI-1 protein.This experiment further wanted to explore whether NgAgo protein and IntI-1 protein interact directly or indirectly by binding to genomic DNA at the same time.This experiment proved that DNase I can digest genomic DNA.DNase I was added to the supernatant of the crushed samples co-expressed by NgAgo protein and inti-1 protein and treated for 1h,and the FLAG antibody resin was used for fishing experiments.It was found that NgAgo protein and IntI-1 protein still interacted,indicating that NgAgo protein and IntI-1 protein were not affected by DNase I,implying that the interaction may be direct.2)In order to further analyze the role of the four domains and enzyme activities of the NgAgo protein in the interaction between the NgAgo protein and the IntI-1 protein,the expression plasmid of different NgAgo domain proteins fused with FLAG tags were first constructed:PET2 8 a-Ng Ago A-FL AG(N-terminal),PET28a-NgAgoB-FLAG(PAZ-MID-PIWI domain),PET28a-NgAgoD-FLAG(MID-PIWI domain),PET28a-NgAgoD-M-FLAG(MID-PIWI domain of enzyme active site mutation),PET28a-NgAgoE-FLAG(MID domain),PET28a-NgAgoF-FLAG(PIWI domain),and the IntI-1 protein expression plasmid fused with GST tag-PGEX-6P-1-IntI-1-GST was constructed.Different NgAgo domain protein expression plasmids and IntI-1 protein expression plasmids were co-transformed into BL21 strains and induced expression,FLAG antibody resin and GST resin were used to conduct fishing experiments on the supernatant of broken samples of co-expressed strains.Fishing results showed that IntI-1 protein interacted with NgAgoB,NgAgoD,and NgAgoF proteins,but not with NgAgoA protein and NgAgoE protein,indicating that the PIWI domain is a key domain for the interaction between NgAgo protein and IntI-1 protein;It also showed that the NgAgoD-M protein also interacted with the IntI-1 protein,and the NgAgoD-M protein is a nuclease-free NgAgo truncated protein with the enzyme active site of the PIWI domain mutated,indicating that the nuclease activity of the NgAgo protein didn’t affect the Interaction of NgAgo protein and IntI-1 protein.In addition,in order to further verify that the interaction between the NgAgo truncated protein and the IntI-1 protein didn’t depend on genomic DNA,the NgAgoD protein was selected in this experiment.The result of GST resin fishing experiment showed that the interaction of NgAgoD protein and IntI-1 protein was also not affected by Dnase I,the interaction of NgAgoD protein and IntI-1 protein may also be direct.3)It was known that IntI-1 protein has the ability to bind target DNA sequence.In order to further explore the influence of NgAgoD-M protein on the binding of IntI-1 protein to target DNA sequence,this experiment synthesized the known target DNA sequence of IntI-1 protein—attI-dsDNA fragment,irrelevant DNA sequence—aadAI-attc dsDNA fragment,and purified recombinant IntI-1 protein and NgAgoD-M protein in vitro.Gel migration experiment verified that IntI-1 protein binded to target DNA sequence and didn’t bind to irrelevant DNA sequence,Proved the feasibility of the experimental condition;further gel migration experimental result showed that NgAgoD-M protein had the ability to inhibit the binding of IntI-1 protein to the target DNA sequence.4)Because the integrase IntI-1 protein and λInt protein belonged to the tyrosine recombinase family,this study added a certain titer of lambda phage to K12 bacteria strains of expressing NgAgo protein or NgAgoD protein,and measured growth curves of these strains and counted the number of plaques formed by these strains,to explore the effect of NgAgo protein on the efficiency of homologous recombination mediated by the binding of λInt protein to target DNA sequence in X phage.It was known that the physiological process of lambda phage infecting host bacteria to some extent represents the efficiency of XInt protein-mediated DNA recombination.After the addition of lambda phage,results of these growth curves showed that there was no significant difference in the growth cycle between the K12 strain group of NgAgo protein,NgAgoD protein and the control group.It was found that there was no statistical difference in the number of plaques between the K12 strain group of expressing NgAgo protein and NgAgoD protein compared with the control group.Therefore,this experiment failed to determine that NgAgo protein and NgAgoD protein can affect the efficiency of DNA recombination mediated by the binding of λInt protein to the target DNA sequence,indicated that NgAgo protein and λInt protein may not interact,but only interact with IntI-1 protein.In general,results of this study found that NgAgo protein can interact with IntI-1 protein through the PIWI domain,and NgAgo protein had the ability to inhibit IntI-1 protein binding to target DNA sequence.