| The LPS structure on the surface of Pseudomonas aeruginosa is the recipient of Pseudomonas aeruginosa bacteriophage,which begins with the adsorption to bacterial surface receptors.Pseudomonas aeruginosa bacteriophage K8 recognizes the O-specific antigen structure on the LPS surface using LPS as a receptor.In this study,we screened a spontaneous mutant of bacteriophage K8 and found a phage with a wider host range and stronger infectivity,which we named GK8.Studies on the general properties of bacteriophages indicate that the stability of the phage GK8 mutant strain is consistent with that of the wild type and is more tolerant to temperature and pH.In order to identify the mutation site of the mutant,we carried out whole genome sequencing of the mutant phage.The genome alignment showed that the base A of the 43851th on the phage K8 genome changed to the base G and the amino acids changed correspondingly.Acid T was mutated to alanine A and the rest were consistent with the wild-type bacteriophage K8,a bioinformatic analysis of the C-terminus of a putative protein,Gp075,from which we hypothesized that the hypothetical protein was associated with phage adsorption,Probably a new phage ligand protein.Receptor analysis showed that GK8 was able to infect OSA deficient strains but not to the core oligosaccharide-deficient strain,suggesting that the receptor recognized by GK8 may be the core oligosaccharide.Phage blocking experiments revealed that the core oligosaccharide of LPS blocked the phage ligand to reduce the ability of GK8 to infect without affecting the ability of K8 to infect,confirming that the GP075 mutant interacts with the core oligosaccharide.Adsorption rate experiments showed that GK8 had different adsorption rates for different host cells,and the highest PAK of wild-type strain was even higher than that of K8.Therefore,it was speculated that GK8 could recognize the core oligosaccharides and OSA of lipopolysaccharide simultaneously.The frequency of spontaneous mutation of phage was also relatively high due to the short cycle of phage infection and the rapid propagation rate.The results showed that phage K8 directed against specific receptor-deficient mutants with spontaneous mutation rates between 4.2 × 10-8 and 2.3×10-7.In order to verify the mutation frequency of gp075,we constructed a mutated library of bacteriophage K8 and sequenced it by amplicon to determine the mutation site of the gene gp075.Among the 153th sequences,we found that 84.2%of amino acids inserted 7 amino acids,5.9%of the amino acids inserted into the 105th position of 14 amino acids,0.7%of amino acids inserted into the 105th position of 21 amino acids,and 9.2%of the point mutations were mutated to GK8,239th amino acid changed from Thr to Ala.Our experimental data show that phage can recognize new receptors on host bacteria by mutation and form new ligand proteins.It is confirmed that there is still a huge genetic diversity in the same phage subpopulation,and the response to host bacteria Change,compete with each other and coevolve.The bacteriophage GK8 has the characteristics of a wide host and can infect many LPS-deficient mutants,and has good phage therapeutic potential.Experiments show that the spontaneous mutation rate of PAK tolerant K8 ranges from 1.0 ×10-6 to 5.0 ×10-6,and is tolerant to GK8 Of spontaneous mutation rates of 1.34×10-9 to 4.91 ×10-9,under the same conditions,GK8 can greatly reduce the spontaneous mutation rate of PAK,therefore,the phage GK8 more potential for the treatment of bacterial infection. |
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