Research On T4 Phage Covalent Modification Of Tolerating Bacterial Crispr And Thoeris Defense System

Drug-resistant bacteria poses a huge threat to animal husbandry and public health.The defense system of bacteria can restrict phage infection,phages have also evolved anti-mechanisms that can tolerate bacterial defense systems,Then the research on the defense mechanism and the anti-mechanism of them has become the key to the development of phage therapy.The purpose of this study is to use T4 phage as a model to explore the role of DNA covalent modification of phage in tolerating the defense system of bacteria,therepy providing theoretical guidance for phage therapy.The details are as follows:1.DNA covalent modification of T4 genome can tolerate different types of CRISPR systemsCRISPR systems are both bacterial defense systems that rely on nuclease activity.Until now,6 types（I-VI）of CRISPR system have now been found,in which types I,II and V target DNA and other types of targeting RNA.Previous studies have shown that the hydroxymethylated glycosylation of cytosine（ghm C）of the T4 genome can help T4 phages tolerate type I and type II CRISPR systems.In order to explore whether ghm C of the WT T4 genome can tolerate the V-type CRISPR system,a number of type-V CRISPR system plasmids targeting the T4 genome were constructed,transformed into E.coli B834,which were infected by the WT T4 and cytosine-modified defective T4 phage（T4（C））infection,respectively.The results showed that WT T4 produced significantly more plaques than T4（C）.This suggests that the ghm C modification can tolerate the V-type CRISPR system.In order to further explore the tolerance of ghm C modification to different types of CRISPR systems,type II and type V CRISPR systems targeting or cleaving the same region of WT T4 were constructed,transformed into E.coli,which were infected by WT T4 respectively.The results showed that the number of plaques formed on the surface of E.coli dishes containing the type II CRISPR system was higher.This suggests that ghm C modifications are more tolerant to the type II CRISPR system,The reason for this phenomenon may be that the Cas protein of V-type CRISPR has a more open structure.Studies have shown that the number of specific spacers of the CRISPR system is positively correlated with the ability of the CRISPR system to resist phage infection.In order to explore whether a variety of CRISPR complexes targeting different sites can more effectively resist phage infection,type-V CRISPR system plasmids targeting one or two different sites were constructed,transduced into E.coli,which infected by WT T4,respectively.The result showed that fewer plaques formed on the surface of E.coli containing the CRISPR system targeting two different sites.This suggests that multiple CRISPR complexes targeting different sites are more effective to resist phage infection.Previous results in this study suggest that ghm C modifications are resistant to the V-type CRISPR system.To this end,we further explored whether the protospacer or PAM sequence in the progeny phage of WT T4 would change under the pressure of the V-type CRISPR system.WT T4 was subcultured under the same pressure of the V-type CRISPR system,The phage plaques of each generation were picked for PCR amplification and sequencing.The result showed that the proportion of progeny phages with mutations in Protospacer or PAM continued to increase under the pressure of continued V-type CRISPR systems.This suggests that the CRISPR system is a double-edged sword that drives the rapid evolution of phages while defending against phage infection.2.DNA covalent modification of T4 genome can tolerate Thoeris systemThe bacterial Thoeris defense system consists of Ths B and Ths A proteins,of which Ths B contains a TIR domain,which is widely present in animal and plant pathogen-associated molecular pattern receptors and plays a key role in the recognition and response of animals and plants to pathogens.Studies have shown that the Ths B protein of the Thoeris system of Bacillus cereus can recognize phage infection and activate the antiviral activity of the downstream Ths A protein,but the mechanism by which Ths B recognizes phage is unknown.When we infected E.coli Nissle 1917 with WT T4 and T4（C）,respectively;we found that only WT T4 could complete the replication cycle and produce progeny viruses,indicating that this strain can distinguish the genomes of the two T4 phages.Analysis of its genome sequence,we found that the Thoeris system may be the main reason.In order to explore the recognition and defence mechanism of E.coli Thoeris system,we cloned the Thoeris system of Nissle 1917,which contains tcp C,RS11255and RS11260 proteins,in which both tcp C and RS11255 contain a TIR domain,which is similar to Ths B of the Bacillus Thoeris system.It was further confirmed that only WT T4 could form plaques in the strains containing the Thoeris system using E.coli B834 and DH10B,indicating that the Thoeris system could protect against T4（C）,but not WT T4.No nuclease domain was revealed by the functional domain analysis of the Thoeris system,indicating that the system does not differentiate WT T4 and T4（C）genomes by cleaving phage DNA,suggesting that the T4 phage may escape the recognition of the Thoeris system through ghm C modification.To explore the defense mechanism of the E.coli Thoeris system,we constructed the Thoeris system lacking tcp C,RS11255 and RS11260 genes,respectively,transformed into E.coli DH10B,infected with T4（C）,and analyzed the defense ability of the above Thoeris system against T4（C）by plaque assay.In order to further explore the escape ability of genome modification to the Thoeris system,T4（α-ghm C）phage containing only cytosineα-glycosylation modification,T4（β-ghm C）containing only cytosineβ-glycosylation modification,T4（hm C）phage containing only cytosine hydroxymethylation modification were constructed by CRISPR gene editing technology.The defense efficiency of the Thoeris system against T4 phages with different degrees of modification was analyzed by plaque experiments.The results showed that the plaque formation efficiency of T4（C）and T4（hm C）was significantly lower than that of T4（α-ghm C）and T4（β-ghm C）,indicating that glycosylation is the key for T4 to escape the Thoeris system,and phage DNA modified by hydroxymethylation can not escape the Thoeris system.Through bioinformatics analysis,it was found that the TIR domain of RS11255and the NAD binding domain of RS11260 protein contain conserved enzyme catalytic center,and the amino terminus of RS11260 protein contains a transmembrane domain.We found that the Thoeris system lost its ability to defend against T4（C）infection after mutating the conserved catalytic site of RS11255,RS11260 and deleting the transmembrane domain of the RS11260 protein.This means that the TIR domain is necessary for the antiviral function of the Thoeris system,and the RS11260 protein needs to be on the cell membrane for antiviral function.Through the phage lysing bacterial curve experiment,it was found that the E.coli Thoeris system resisted T4（C）infection by inducing bacterial abortive infection.These results suggest that the Thoeris system recognizes phage infection through RS11255,and uses the RS11260 protein to induce bacterial abortion to play an antiviral function.In order to further explore whether the antiviral activity of RS11260 requires the activation of the RS11255 protein,we used arabinose-induced expression vectors to construct the RS11260 protein expression vector and the RS11260 and RS11255 protein co-expression vectors,which were transformed into E.coli DH10B respectively.T4（C）and T4（hm C）infection experiments showed that only co-expression of RS11260 and RS11255 proteins could resist T4（C）and T4（hm C）infection,indicating that the antiviral activity of RS11260 protein requires the activation of RS11255 protein.In order to explore how RS11255 protein activates the antiviral activity of RS11260protein,we found that RS11255 protein may degrade NAD molecules into ADPR molecules by chromatography-mass spectrometry（LC-MS）technology,which indicates that ADPR molecules may be responsible for the antiviral activity of RS11260protein.activator.The above results indicates that RS11255 protein degrades NAD molecules into ADPR molecules after recognizing phage DNA,and the generated ADPR molecules activate the antiviral activity of the downstream RS11260 protein,which ultimately leads to bacterial abortion.3.Development of Novel Phage Gene Editing TechnologyGiven that the type V CRISPR system is more efficient in cutting the ghm C genome,this system was used in this study to develop a novel and efficient phage genome editing technology.The system contains a plasmid expressing type-V CRISPR-Cas and a donor plasmid for homologous recombination,wherein the expressed CRISPR-Cas complex is responsible for cleaving the phage genome,and the donor plasmid undergoes homologous recombination with the fragmented phage genomic DNA,Thereby the target DNA is recombined into the phage genome.In order to evaluate the editing efficiency of this method,we first selected theα-glycosylation gene andβ-glycosylation gene with a fragment length of 1²kb,and obtained T4（α-ghm C）,T4（β-ghm C）和T4（hm C）through homologous recombination,respectively,the recombination efficiency is about 10^-310^-4,and there is no wild-type T4 phage background.To further evaluate the ability of the method to edit long DNA fragments,we attempted to delete the¹0.8kb fragment between genes 39 and 56 of T4 phage,which encodes a non-essential gene,and the deletion did not affect phage proliferation.The results show that the editing system can efficiently obtain recombinant phage,the recombination efficiency is 10^-3,and there is also no wild-type T4 phage background.To evaluate the efficiency of this technology for editing other phages,we performed site-directed mutagenesis on genes 4,5,9,and 19 of the T7genome,respectively,and all of them were able to efficiently obtain the expected mutants with a recombination efficiency of 10^-310^-5,and produced little T7 phage background.The above results show that the phage gene editing technology based on the type-V CRISPR system is an effective gene editing tool,which can efficiently perform point mutation and gene deletion on the bacterial genome.In summary,this study reveals that T4 phage is tolerant to bacterial CRISPR and Thoeris defense systems through DNA covalent modification;bacteria enhance their ability to cleave covalent modified DNA by expressing Cas complexes targeting different sites;based on V-type The CRISPR system,we have successfully developed a novel phage gene editing technology.