Isolation,identification,whole Genome Sequencing Of Bacteriophage Against Pseudomonas Aeruginosa Isolated From Forest Musk Deer,and Prokaryotic Expression Of Slt56 Protein

Forest musk deer(Moschus berezovskii)is a first-class protected animal in China.The musk secreted by adult male musk deer is very precious and widely used in medicine and perfume.With the indiscriminate hunting and destruction of the ecological environment,the wild musk deer resources are on the verge of exhaustion.Artificial breeding forest musk deer is a relatively effective way to protect forest musk deer.Pseudomonas aeruginosa(PA)is a gram-negative bacterium.As a common opportunistic pathogen,the suppurative diseases caused by PA cause more than half of the deaths in captive forest musk deer.As a potential antibacterial factor,bacteriophages can infect and replicate in bacteria.Its products such as endolysin and lytic glycosyltransferase(LT)also have great application value in the field of anti-bacterial infection.Artificial breeding of forest musk deer has a broad prospect,but the suppurative diseases caused by PA have seriously hindered the development of artificial breeding of forest musk deer.The unreasonable use of antibiotics also leads to severe drug resistance in PA,making its prevention and treatment quite difficult.Therefore,there is an urgent need to find and develop new antibacterial drugs.In this study,a PA strain isolated from the lungs of dead forest musk deer was used as the host,and a virulent bacteriophage was isolated from the daily drinking water of captive forest musk deer in Sichuan Institute of Musk Deer Breeding,which was named v B＿PaeM＿PAMD02.The biological characteristics of the bacteriophage were studied,its morphology was observed by scanning electron microscope,and the bacteriostatic test of v B＿PaeM＿PAMD02 in vivo and in vitro against PA was carried out.The whole genome sequencing and analysis of v B＿PaeM＿PAMD02 was carried out.Bioinformatics analysis and prokaryotic expression of the soluble glycosyltransferase(Slt)protein Slt56 encoded by gp56 gene were performed,and the activity of slt56 in vitro was preliminarily verified.The main test results are as follows:1.A virulent bacteriophage named v B＿PaeM＿PAMD02 was isolated.After purification,the contour of the bacteriophage plaque is clear,without halo ring,and the original titer is 1.59×10~9.Electron microscopic observation shows v B＿PaeM＿PAMD02 has a typical icosahedral head and retractable tail structure of the Myoviridae.The best multiple of infection(MOI)test showed that its best MOI was 0.1.The one-step growth curve test showed that the bacteriophage had an incubation period of 40 min,a lysis period of 60 min,and an outbreak of 65.8 PFU/cell.The thermal stability test showed that the bacteriophage had good stability at higher temperature,its potency was higher than 37℃at 50℃,but its potency decreased significantly at higher temperature.The optimum p H showed that the phage had good tolerance to weak acid environment,but its titer decreased significantly in weak alkaline environment.v B＿PaeM＿PAMD02 is not sensitive to chloroform and ultraviolet rays,and its combined use with EDTA can effectively improve the antibacterial effect.v B＿PaeM＿PAMD02 showed good bacteriostatic effects in vivo bacteriostatic tests in mice.2.Whole genome sequencing display v B＿PaeM＿PAMD02 has a genome size of 66 264 bp and a GC content of 55.59%.It has 92 open reading frames(ORFs)and no non coding RNA.ANI analysis shows that v B＿PaeM＿PAMD02 belongs to PB1 like bacteriophage.In addition to the endolysin gene and the Slt protein gene,v B＿PaeM＿PAMD02 also has a variety of catalytic enzyme genes related to life cycle and cleavage activity,with no virulence genes or lysogen genes.Evolutionary tree analysis based on major capsid proteins and large subunits of terminal enzymes,v B＿PaeM＿PAMD02 belongs to the genus Pbunavirus under the Myoviridae family.3.Analysis results of protein structure and physicochemical properties showed that the Slt56protein had no signal peptide and contains a single soluble lytic transglycosylase domain.In order to explore the activity of this domain in vitro,prokaryotic expression of the Slt56protein was performed,the expression conditions were optimized,and the Slt56 was purified.In this article,the in vitro activity was preliminarily verified by plate counting method and plate diffusion method.The preliminary validation results of in vitro activity showed that although Slt56 protein does not have a separate antibacterial ability,when used together with v B＿PaeM＿PAMD02,it can significantly improve the efficiency of v B＿PaeM＿PAMD02infection and bacterial lysis.In summary,this study successfully isolated and identified the first Pseudomonas aeruginosa bacteriophage from forest musk deer.The research and analysis on its in vitro and in vivo antibacterial activity,as well as genome-wide related biological information were conducted.And bioinformatics analysis,prokaryotic expression,and preliminary validation of its Slt56protein in vitro activity were carried out.All the studies provide a theoretical basis for the subsequent application of bacteriophages and their related proteins,as well as for the clinical diagnosis and treatment of purulent diseases in forest musk deer.