Genetic Polymorphism Drives Coevolution Between Bacteria And Bacteriophages

In recent years,the emergence and spread of bacterial resistance have brought great challenges to clinical anti-infective therapy.The threat of super drug-resistant bacteria to human health has been paid more and more attention,and phage therapy,which can treat antibiotic-resistant bacterial infection,has attracted wide attention again.However,in phage therapy,bacteria can easily develop resistance to phages,which significantly blocked the development of phage therapy.Therefore,to explore the mechanism of bacteria rapidly developing strong phage resistance and to overcome the tolerance of bacteria to phage has become the primary problem in the field of bacteriophage treatment of bacterial infection.In theory,bacteriophages,unlike antibiotics,are viruses that can evolve to regain their infectivity and lethality to bacteria that have acquired phage resistance.Based on the characteristics of phage and researches on the co-evolutionary mechanisms of bacteria and bacteriophages,and to explore and overcome the problem of rapidly developed phage resistance,the current studies were carried out using the next generation sequencing(NGS)technology to further explore the mechanism of changes in susceptibility of both sides in the coevolutionary system of bacteria and bacteriophages.In the first part of the thesis,Staphylococcus aureus AB91118 and its lytic phage LQ7 were studied as a model system to analyze the mechanism of bacteria and phage coevolution.A mutant strain named R1-3-1 resistant to the ancestral phage LQ7 was isolated,and then phages experimentally evolved from LQ7 were able to kill R1-3-1.The bacterial genome analysis revealed that there was only one point mutation in the non-coding region on the genome of mutant R1-3-1,which was difficult to explain the reason of the tolerance of the mutant to phages.Therefore,this study innovatively introduced the concept of quasi species to the study of bacteria and phage coevolution,and independently developed a method based on the minor allele identification to determine the genetic polymorphism sites.Using this method,it was found that there were 57 unique genetic polymorphisms on the genome of R1-3-1,which were found on 38 functional genes involved in energy metabolism.It was speculated that R1-3-1 might improve its own energy metabolism to acquire resistance to phage LQ7.Furthermore,chloramphenicol,which can inhibit bacterial energy metabolism,was selected in combination with LQ7,and it was found that sublethal levels of chloramphenicol combined with LQ7 phage could effectively inhibit the growth of R1-3-1,which proved the above speculation.In the case of phages,it was also demonstrated that genetic polymorphism assisted their evolution and restored their bactericidal ability against mutant R1-3-1.In addition,chloramphenicol combined with evolved phages can help overcome the development of bacterial tolerance to a certain extent.Based on the above results,this part of the study found and proved that Staphylococcus aureus and its lytic phage exists genetic polymorphism with minor alleles,and genetic polymorphism is one of the important factors affecting their coevolution.In the second part,based on the findings in the first part,public data mining was conducted to verify that the genetic polymorphism was not an accidental phenomenon in bacteria and phage coevolution.By the means of data mining,high-throughput sequencing data of the coevolution experiment of Enterococcus faecalis TX1330 and its lytic phage Ef V12-phi 1 from the SRA database of NCBI were obtained.Using the method developed in the first part,it was found that there were genetic polymorphisms in the genomes of both bacteria and phages in the coevolution systems.The evolved Enterococcus faecalis specific genetic polymorphisms were found in functional genes involved in catalytic activity changes.Phages also have genetic polymorphisms in the gene encoding tail fiber protein.In addition,the highthroughput sequencing data of four isolated bacterial genomes also indicated genetic polymorphisms existing in bacterial genomes.These results firstly demonstrate that genetic polymorphism is common in the co-evolutionary process of bacteria and bacteriophages and secondly extend our understanding of bacteria and other prokaryotes at the population genome level.The third part of this thesis is based on the analysis of the first two parts,proving that genetic polymorphism also exists in the adaptive evolution of phages under abiotic stress.Three phages,Wc4,CX5 and P-PSG-11,which can lyse Pectobacterium carotovorum subsp carotovorum,Pectobacterium atrosepticum,and Ralstonia solanacearum effectively,respectively,were adapted to high temperature evolution,and three more stable mutant phage strains were screened out.Highthroughput sequencing and analysis of phage genomes before and after adaptive evolution revealed that there were genetic polymorphisms in all six phage genomes.These genetic polymorphic sites were found in functional genes such as genes encoding tail tubular protein,DNA helicase and DNA polymerase,which maintain the structural stability of phages and are involved in genomic DNA replication.It is proved that in addition to mutation,genetic polymorphism also provides a genetic basis for the adaptive evolution of phages in response to adversity.The results from the above three parts of the thesis prove that genetic polymorphism is prevalent in bacteria and phages and drives the coevolution of bacteria and phages.More importantly,in this thesis it was proposed for the first time that the functional genes of specific genetic polymorphisms can be used to predict the changes in microbial physiological functions during bacterial evolution.And a new therapeutic method of targeting effective drug based on the predicted results combined with phage evolution was also proposed.It can overcome the bacterial tolerance to a certain extent and improve the effectiveness of phage therapy.These findings not only provide an important theoretical basis for rational application of phage therapy but also have practical guiding significance for clinical application of phage.