Efficient Formation Of Enterobacterial Ghosts Mediated By Phage ID52 Lysis Protein E

Bacterial ghosts（BGs）,the empty bacterial shells without cytoplasmic contents such as genome and protein,retained the complete morphology and surface antigen components of natural bacteria,which can induce strong mucosal immune response,humoral response and cell immune response.On the one hand,BGs retained the immune stimulation complexes on the bacterial surface and had inherent adjuvant properties,makng them potential to be new effective vaccine candidates.On the other hand,BGs had a very strong loading capacity and can be used as good delivery carriers for carrying substances such as exogenous drugs,DNA and protein antigens.Escherichia coli Nissle 1917（EcN）is probiotics that can be used to regulate intestinal flora,alleviate inflammation and targeted therapy for tumors.Studies have shown that EcN BGs can be used as targeted delivery carriers for anti-tumor drugs and has a good application prospect in targeted therapy of tumors.Salmonella typhimurium（ST）is a kind of Gram-negative food-borne pathogens that can cause zoonotic diseases.Studies have shown that ST BGs can be used as animal vaccines or antigens carrier vaccines to effectively protect animals from the infection of pathogens.However,the yield of traditional BGs preparation methods was very low,which limited the large-scale production and industrial application of BGs.In order to overcome this problem,many scholars have studiedφX174-E,but the yield of BGs was still far from the demand of large-scale production and application.The BGs formation method mediated byφX174-E has reached the bottleneck period.In this study,after preliminary literature investigation and preliminary experimental screening,it was found that E.coli phage ID52 lysis protein ID52-E can effectively lysis E.coli,but no relevant studies have applied it to the preparation of BGs.In view of this,the following experiments were carried out on lysis protein ID52-E in this study.Firstly,E.coli BL21（DE3）was used as host bacteria,and the lysis protein ID52-E of E.coli phage ID52 was recombinant expressed for the first time in order to study the feasibility of its application in producing BGs.The detection of the lysis curve,cytoplasmic contents leakage,BGs formation efficiency,and proteins expression levels showed that the cleavage efficiency of ID52-E was significantly better than that ofφX174-E,and the arabinose inducible promoter significantly increased the lysis activity of ID52-E.The high-quality empty shell BGs with complete structure and lysis pores were observed by Scanning electron microscopy（SEM）and transmission electron microscopy（TEM）,and there was no significant difference in the morphology of the BGs formed by the two kinds of lysis proteins.It can be concluded that arabinose induced lysis protein ID52-E lysis system was the best efficient preparation system of BGs.When the initial induced OD₆₀₀ was about 2.0（1.3×10⁹ CFU/mL）,0.5 mg/mL L-arabinose was added to induce ID52-E expression,and the lowest OD₆₀₀ can decrease to 0.3～0.4,which significantly increased BGs production.Secondly,λRed homologous recombination technique was used to construct EcNΔaraBAD::FRT engineered strain and STΔaraBAD::FRT engineered strain.Then,the above efficient preparation system was introduced into EcN and ST bacterial ghosts preparation,and the initial induced OD₆₀₀ was up to 2.5,which significantly increased the yield of BGs.SEM and TEM observed that EcN BGs and ST BGs retained the cell morphology and cell membrane integrity without cytoplasmic contents.Finally,in this study,site-directed mutation of C-terminal cysteine of lysis protein ID52-E was conducted to investigate whether lysis protein ID52-E also existed in the form of polymer when forming lysis pore and whether C-terminal cysteine was involved in the formation of lysis protein ID52-E polymer.The mechanism of lysis protein ID52-E of E.coli phage ID52 was preliminarily discussed to explain the potential reason for the difference of lysis activity betweenφX174-E and ID52-E.The results indicated that cysteine at the C-terminal is involved in the formation of the polymer of ID52-E,and cysteine at position 61th was the key site of lysis activity.In summary,in this study,E.coli phage ID52 lysis protein ID52-E was recombinant expressed for the first time and applied to the preparation of BGs.By optimizing initial induction OD₆₀₀ and promoters,we screened out the best BGs preparation system realizing the efficient preparation of E.coli BL21（DE3）BGs,EcN BGs and ST BGs,which provided a new method for the efficient preparation of BGs.Meanwhile,the mechanism of lysis protein ID52-E was preliminarily discussed by site-directed mutation of C-terminal cysteine.