| Antibiotics have been widely used in animal husbandry production and disease prevention and control,which has induced many pathogenic bacteria to become drug-resistant."Superbugs"(multi-drug and high levels of drug resistance)have made the problem become more complex and severe.The problem of bacterial resistance is getting worse,which has seriously threatened public health and human resources of antibiotics.Phage lyase can efficiently,rapidly and specifically lyse drug-resistant pathogens without affecting the symbiotic flora and it is not easy for bacteria to develop tolerance to lyase.Lyase lyses bacteria by catalyzing peptidoglycan since it is the skeleton of the cell wall.Gram-positive bacteria can be directly lysed by lyase because peptidoglycan lie in the outer layer of the cell wall so that it is easy to prepare gram-positive bacteriaphage lyase.At the same time the outlayer of gram-negative bacteria is tight out membrane protein and only allows the passage of small molecules.And peptidoglycan of gram-negative bacteria is in the periplasmic space of the cell and the synthetic lyase cannot lyse the gram-negative bacteria directly form the outside and then hinder the development of lyase.This paper focus on this problem and want to construct genetic engineering lyase to explore how the lyase can cross the outer membrane and kill E.coli which is a kind of gram-negative bacteria.Bioinformatics analysis was conducted on macromolecular proteins Colicin A,Lys AB2,SPN9 CC,Lys PA26 and D8,which can cross the outer membrane of gram-negative bacteria in nature.Sequences were put in DNAstar software for homology analysis,and their homology was found to be unrelated.Thus it was speculated that their transmembrane function was independent of the amino acid sequence.Their transmembrane proteins,physical and chemical properties and homologous modeling and tertiary structure prediction were analyzed by online software TMHMM,Ex PASy and SWISS-MODEL separately.What was found in common among these three proteins was that the proportion of positively charged amino acids and hydrophobic amino acids was higher in some regions or at the end.So we hypothesized that hydrophobic amino acids and positively charged amino acids could help lyase to cross the outer membrane of gram-negative bacteria.To verify the hypothesis,the effect of hydrophobic amino acids on the permeability of lyase through the extracellular membrane was first studied.The Lysep3 studies in the previous work could not lyse E.coli from outside the bacteria.Based on the Lysep3,five fusion proteins Lysep3-3,Lysep3-5,Lysep3-7,Lysep3-12 a and Lysep3-12 b with hydrophobic index of 1.711,2.311,2.889,4.089 and 4.089,respectively,were constructed by the C-terminal modification with different hydrophobic amino acids Ile,Val,Leu,Phe and Ala.Among them,Lysep3-12 a and Lysep3-12 b had the same hydrophobic index but different amino acids sequences.These 5 proteins were expressed and purified by prokaryotic expression system.The data showed that they had the highest lytic activity at p H 5 and the concentration of 1.75 ?g/m L.EDTA could enhance the bactericidal effect of fusion lyase.Lysep3-12 a and lysep3-12 b had the same lytic ability,which verified the hypothesis that the lyase permeating the outer membrane was independent of the sequence.The results indicated that the hydrophobic amino acids at the C-terminal of lyase could help phage lyase to lyse E.coli from outside of the bacteria.Within a certain range,the ability of lyase to lyse bacteria was positively correlated with the number of terminal hydrophobic amino acids.Next,5 cationic amino acids KRKRK were modified at the C-terminal of Lysep3 to build a fusion lyase Lysep3-5aa,10 cationic amino acids KRKRKRKRKR were modified at the C-terminal of Lysep3 to build a fusion lyase Lysep3-10 aa and 15 cationic amino acids KRKRKRKRKRKRKRK were modified to build the lyase Lysep3-15 aa.Their isoelectric points were 11.37,12.31,12.48,respectively.A mixture of hydrophobic and cationic amino acids was constructed at the end of Lysep3 as control which named Mix.These fusion lyases were expressed by prokaryotic expression system and purified.The results showed that they had different bactericidal properties,and the bactericidal ability was positively correlated with the number of positive electric amino acids modified on C-terminal.Among them,the fusion lyase Lysep3-15 aa had better bactericidal activity and can lyse salmonella,shigella and chloragrobacterium.According to the above experimental results,Lysep3,the N-terminal of Colicin and BR gene sequences had a combination modification.According to the results,a fusion lyase Colicin-Lysep3 with N-terminal cationic amino acids and hydrophobic amino acids was designed.Colicin-Lysep3 was expressed and purified by prokaryotic expression system.Under the electron microscope,the cell wall of E.coli was lysed,cell contents discharged,and some cells formed bacterial ghosts.Colicin-Lysep3 showed the strongest bactericidal performance at p H 5.Colicin-Lysep3 showed better bactericidal property also in vivo experiments.The number of labeled E.coli in the anterior of small intestine of mice treated with Colicin-Lysep3 was 10.70% fewer than that in the control group averagely.And the number of in the posterior segment of the small intestine was reduced by 3.23%,and the cecum by 2.63%.Further studies found the fusion protein firstly bind to the E.coli at the recognition area.And then,with the help of N-terminal charge and hydrophobic peptidem,lyase could contact with peptidoglycan across the outer membrane and catalyze the glycosidic bond between acetylparietal acid and N-acetylglucosamine.As a result,the cell wall skeleton was broken and under the action of osmotic pressure,the cell contents leaked out and cells died.If Colicin-Lysep3 is directly added in feed,it would be costly and impractical.In this study,the method of traceless secondary homologous recombination was used to replace the Spa0 gene of a Bacillus subtilis of good stress resistance with Colicin-Lysep3 gene to construct the engineering strain ?B.subtilis.?B.subtilis expressed Colicin-Lysep3 and secreted it to the outside of bacteria,with the function of killing E.coli in vivo and in vitro.In conclusion,the modification of hydrophobic amino acid and charged amino acid at the C-terminal of the phage lyase could help lyase to lyse gram-negative bacteria from the outside.Within a certain range,the ability of lyase to penetrate the outer membrane was positively correlated with the number of terminal hydrophobic amino acids and charged amino acids.This discovery provides a theoretical basis for the artificial synthesis of phage lyase which can lyse gram-negative bacteria in the future. |
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