Analysis Of Genetic Evolution Of Staphylococcal Phage Genome And Detection Of Its Lysin Activity

Staphylococcus,mainly staphylococcus aureus,play important roles in human and animal infections.However,antibiotics treatment for Staphylococcus infection is restricted by the emergence of drug resistance,thus,more effective preventative or treatment methods are desperately needed.As a unique bacterial virus,staphylococcal phages can parasitize staphylococcus and kill staphylococcus,their lysins can degrade the bacterial peptidoglycan,which makes them promising antibacterials for future use.Therefore,the researches on phages and their lysins are of great significance to the prevention and treatment of staphylococcus infections.(1)The genomic information of staphylococcal phages,mainly staphylococcus aureus phages,were retrieved from the NCBI database.Then the relationship between phage nucleotide usage and phage phenotype,as well as its codon usage preference,overall codon usage pattern,parity rule 2 analyses,effective number of codons and GC3 content were analyzed by bioinformatics methods.The results confirmed that there is a significant relationship between the staphylococcal phage nucleotides usage and the phage phenotype.In addition,there are significant differences in codon usage,parity preference,effective number of codons and GC3 content of staphylococcal phage among different species.The staphylococcal phage genomes were affected by genetic evolution and mutation.(2)Phage vB?SauS?SAP3 was isolated from sweage by using the double-layer agar methods.Its biological characteristics were analyzed.Phage v B?Sau S?SAP3 can lyse 5 staphylococcus aureus strains and one staphylococcus chromogenes strain,its optimal multiplicity of infection is 1:0.1.When the temperature is higher than 45°C,the phage titer began to drop sharply.Phage v B?Sau S?SAP3 is not resistant to acidic environments but has good tolerance to alkaline environments.Its host spectrum and tolerance to alkaline environments are different from other known staphylococcus aureus phages.Phage v B?Sau S?SAP3 has a genome with a length of 41952 bp and a(G + C)M% content of 35.42%.It contains 65 ORFs,of which,26 encode proteins with known functions and 39 encode proteins with unknown functions.The electron microscope observation revealed that the end of the phage had larger fibrils,which was consistent with the characteristics of staphylococcal phages.Phylogenetic tree analysis showed that phage v B?Sau S?SAP3 is located in the same evolutionary branch as the Bacteriophage EW of the siphoviridae,its main capsid protein gene and lysin gene are located in different branches.Phage v B?Sau S?SAP3 is a new type of the siphoviridae phage with typical genetic evolution characteristics of the siphoviridae.(3)The amidase gene of phage vB?SauS?SAP3 was amplified and ligated into PET-30a(+)vector.The plasmids were transformed into competent cells and the amides(lysin protein)was expressed sucessfully in vitro.The characteristics of the lysin protein were identified in vitro and the therapeutic activity of the lysin protein was verified in vivo.At the same time,the structure of the active domain of the lysin was analyzed,and the activity characteristics of its active domain were verified.The results indicated that the lysin protein is mainly expressed in the inclusion body,can form an obvious antibacterial zone in vitro,and has an inhibitory effect on the existing staphylococcus.Three-dimensional analysis showed that the lysin protein harbors two distinct active domain structures,this two active domains have different antibacterial activities.However,the protective effect of lysin protein and active domain protein is not as good as expected in vivo in an mouse model.