Screening And Preliminary Application Of Bacteriophages That Tolerate E.Coli TIR Defense Systems

The widespread and indiscriminate use of antibiotics has increasingly led to the development of bacterial resistance.Bacteriophages have been considered a powerful weapon for treating antibiotic-resistant bacterial infections.However,the treatment of bacteriophages has been limited by their narrow lytic spectra.As bacteria and bacteriophages constantly co-evolve and battle,bacteria have evolved various defense systems,which we speculate may be responsible for the narrow host range of bacteriophages.Toll-like receptors were initially discovered for their role in the dorsalventral pattern in embryonic development and later found to be related to the immune response in fruit flies.Studies have shown that the TIR domain protein in bacteria determines defense characteristics against invading pathogens and immune specificity.In this study,directed evolution of bacteriophages under the pressure of TIR defense systems in E.coli was used to screen for phages capable of tolerating this system,and a new method of efficiently obtaining broad-spectrum bacteriophages that tolerate TIR defense systems was developed.The study also revealed the mechanisms of host defense and bacteriophage counter-defense,providing a theoretical basis for solving the problem of narrow host ranges in bacteriophage therapy.The main contents of the study are as follows:1.Research on three novel TIR defense systems in E.coliThis study cloned 3 candidate defense systems containing TIR domains based on the laboratory E.coli genome,and successfully constructed plasmids expressing these TIR defense systems.The defense effects of the 3 TIR systems were tested using T4 and T7 phages,and it was found that the 67 system could resist wild-type T7 phages,while all 3 systems could resist wild-type T4 phages to varying degrees.In order to further validate the universal defense effect of these 3 systems,111 phages isolated from the laboratory were used to verify the antiviral activity of their defense systems.The results showed that the 45 system could inhibit 28 phages,the 67 system could inhibit30 phages,and the 70 system could inhibit 15 phages.These results indicate that these3 defense systems have a wide range of defense activities against model phages and phages isolated from the environment.To investigate whether the 3 TIR defense systems defend against phages through an abortion mechanism,this study used a bacterial growth curve test of phage-induced bacterial lysis,adding different doses of T4 phages to E.coli containing the 3 defense systems,and reading the OD600 value of bacterial growth.The results showed that all 3TIR defense systems exerted antiviral effects against T4 phages through abortion of infection mechanism.2.Screening of TIR defense system-tolerant bacteriophagesBacteriophages evolve continuously under the pressure of host defense systems.Theoretically,they can mutate to escape the host defense systems to survive.In this experiment,high doses of phages were used to spot the double-layer agar plate containing E.coli strains with TIR systems.The selected phages were T7 and T4 that were defended by TIR systems 67,70,and 45.After 8,10,and 15 generations of screening under stress,respective T7 and T4 mutants that were tolerant to the defense system were obtained.In order to verify whether the host range of T4 and T7 mutant strains in the tolerant defense system has been expanded,experiments were conducted using 41 pathogenic E.coli strains as host bacteria to test the host range of T4 and T7 mutant strains.We found that T7 phage could not infect 41 pathogenic E.coli strains,but after tolerance,T7 could cause varying degrees of lysis in 15 of them.T4 phage could infect only 4 of the 41 pathogenic E.coli strains,but after tolerance,the T4 mutant strain could infect21 of them.These results confirm that selecting phages tolerant to the defense system can broaden the lysis spectrum of the phages.In order to find the specific genes and mutation sites of phages tolerant to the TIR defense system,we extracted and sequenced the genomes of the phages before and after screening and analysis,and found two mutation sites in gene A of the T7 phage and one mutation site in gene B.Using phage gene editing technology,we obtained single and double-site mutations in gene A and four T7 mutant strains with single-site mutations in gene B of the wild-type phage.Spot tests revealed that only the double-site mutation in gene A could result in escape from the TIR defense system.The laboratory isolated four strains of E.coli bacteriophages from the environment.To broaden their host range,we screened for broad-spectrum phages that were resistant to multiple defense systems.A spot assay was conducted using 13 candidate TIR defense systems constructed by the research group to evaluate the isolated bacteriophages.Based on the defense spectrum results,it was found that EP66 phage and EP133 phage could be defended against by most TIR defense systems.The focus of the experiment was to screen for resistance to multiple defense systems in these two phages.After 25 and 30 generations of repeated screening,two broad-spectrum mutant strains were obtained,namely EP66 mutant which was resistant to six TIR defense systems and EP133 mutant which was resistant to nine TIR defense systems.Further examination of the host range changes in these two phages before and after mutation revealed that compared to the EP66 phage wild type,the EP66 mutant that was resistant to multiple TIR systems could lyse an additional 24 pathogenic strains of E.coli;and compared to the EP133 phage wild type,the EP133 mutant that was resistant to multiple TIR systems could lyse an additional 20 pathogenic strains of E.coli.3.Biological characteristic analysis and antibacterial evaluation of mutant bacteriophages.In order to determine whether there are changes in the biological characteristics of the separated phages before and after mutation,we conducted heat resistance,acid-base resistance,in vitro proliferation,and lytic ability tests.The results showed that the phages EP 133 and EP 66 isolated from the environment showed varying degrees of enhancement in heat resistance,acid-base resistance,and in vitro lytic ability after mutation compared to before mutation.In addition,to test whether the evolved phages have clinical application potential,sterilization effectiveness evaluation was conducted in milk,and the difference in cocktail sterilization effectiveness after mutation compared to before mutation was significant(p < 0.05).In this study,three novel TIR defense systems of E.coli were discovered and verified,and it was confirmed that they all exert a wide range of anti-phage activity through the abortion mechanism.At the same time,by utilizing the directional evolution method of phages under the pressure of TIR defense systems,mutated T4 and T7 and EP66 mutant and EP133 mutant capable of tolerating TIR systems were screened.The lytic spectra of the mutated phages that could tolerate after treatment broadened,which has some application prospects in multi-drug-resistant infection treatment in the livestock production industry.