Study On The In Vitro And In Vivo Antibacterial Activity Of Endolysin Ply0643,its Chimeric,and Screening Of Phenanthridinone Compounds With Antibacterial Activity

There are common zoonotic pathogens in Streptococcus,such as Streptococcus agalactiae,and Streptococcus suis.The drug-resistant bacteria pose a greater threat to public health safety and animal husbandry.Phage endolysin is encoded by the phage genome and can lyse the peptidoglycan layer of bacteria.It can directly dissolve the cell wall outside the bacteria and cause the bacteria to die.The antibacterial spectrum of endolysin is wider than that of bacteriophages,and it is difficult to induce bacterial resistance.It is an excellent substitute for antibiotics and has huge application potential.In previous experiments,we found the potential endolysin Ply0643 from Streptococcus agalactiae GD201008-001 prophage genes,and this protein has been expressed.This article mainly studied the antibacterial activity of endolysin Ply0643 in vivo and in vitro,modified endolysin Ply0643 to obtain lysis activity against Staphylococci,and studied the rapid purification method of endolysin CHAPk using elastin-like polypeptides（ELPs）.Except for new antibacterial agents such as bacteriophages and endolysins,finding new antibacterial agents from compounds is another direction.Existing compound antibacterial agents such as sulfa drugs have been widely used in clinical therapy.Phenanthridone is a compound with a wide range of physiological activities.We screened the antibacterial and low cytotoxic compound HKC20 among 119 synthetic phenanthridones,and studied the mechanism of inhibiting bacterial growth.1 Study on the antibacterial activities of endolysin ply0643 in vitro and in vivoThis study purified the streptococcal endolysin Ply0643.The enzyme’s antibacterial activity was tested at different p H and temperature.The test showed that Ply0643 had antibacterial activity for GBS at p H 5-11 and temperature 4-55°C;0.8 mg Ply0643 is treated by the mouse bacteremia model infected by Streptococcus suis CZ130302 and can protect 80%of mice challenged by intraperitoneal injection of 20 times LD₅₀;After using endolysin to treat the mouse mastitis model infected by Streptococcus agalactiae,the number of bacteria in the mammary gland tissue was reduced by 97.3%compared with the untreated group.The pathological slices of the breast and the transcription level of inflammatory factors showed that the inflammation level of the treatment group was less than that of the treatment group,indicating that endolysin Ply0643 has a therapeutic effect on mastitis caused by Streptococcus agalactiae.Ply0643 has strong stability and good antibacterial effect in vivo and in vitro.2 The expression of chimeric endolysin Ply K0643 and its treatment of mouse pyoderma modelExpression of recombinant protein containing Mur NAc-LAA catalytic domain and Lys M binding domain of Ply0643.By removing the Flg J domain of the endolysin Ply0643,the recombinant protein Ply0643^LMhas the same lytic activity as the full-length Ply0643.the structure of the Staphylococcal endolysin Lys K Domain CHAPk is linked to Ply0643^LMfor recombinant expression,and the resulting chimeric endolysin Ply K0643 has a broader antimicrobial spectrum,including Streptococcus agalactiae,Streptococcus suis,Streptococcus pneumoniae,Streptococcus lactis,Streptococcus equi subsp.zooepidimicus,Staphylococcus aureus and Staphylococcus pseudointermediate.After treatment with Ply K0643 on pyoderma in mice induced by Staphylococcus pseudointermediate,the abscesses in the pyoderma of mice are reduced,and the secretion decreased.Chimeric endolysin Ply K0643 increases the antibacterial spectrum and still maintains good lytic activity in vivo.3 Preparation and expression of endolysin with ELPsIn order to express the endolysin rapidly,we inserted the CHAPk domain sequence of Lys K and endolysin Ply0643^LMinto the recombinant expression vector pET/ELP-I-CAT which containing ELPs andΔI-CM intein,respectively.After inducing expression,we optimize the conditions and use the temperature-sensitive properties of ELPs to perform a reversible phase transition cycle（ITC）for the purification of protein while the self-cleavage properties of the intein releases the target protein.After many trials,Ply0643^LMcannot be expressed by this method.At the same time,ECHAPk obtained soluble expression by ELPs.The ECHAPk obtained by ELPs has a significant lytic activity against MRSA.Using this method to purify the protein not only has high protein purity,no other pyrogenic protein,but also has simpler steps,shorter purification time and low cost,and can be used for large-scale production of endolysin.4 Screening of antibacterial phenanthridone compounds and study on their antibacterial mechanismFive compounds with antibacterial effects were selected from 119 phenanthridone compounds by the drop-plate method;a compound,named HCK20,with low cytotoxicity was screened out in the cytotoxicity test.The antibacterial spectrum of HCK20 was detected by the MIC method,and the result showed that HCK20 has a significant antibacterial effect on Streptococcus when the MIC is between 15μM-60μM.HCK20 have no antibacterial effect against Staphylococcus,Escherichia coli,Klebsiella pneumoniae,Aeromonas hydrophila,Pseudomonas adaceae and Bacillus cereus.By the detection of extracellular soluble protein from bacteria,it can be inferred that one of the antibacterial mechanisms of HCK20 against Streptococci is that it can change the permeability of the streptococcal cell membrane.