| Improved security and increasing medical challenges in the global food system require better monitoring and control to foodborne pathogens.The continuous evolution and prevalence of pan-antibiotic-resistant bacteria,along with the spread of food chain,leads to a sharp rise in the risk of antibiotic resistance,that posing a serious challenge to food safety and medical resources.It is urgent to explore navol bactericidal pipelines.Lysins are proteins released by most mitotic phages during the lytic phase.It has strong peptidoglycan degradation activity and can destroy the cell wall structure immediately after contact with host bacteria,thus killing bacteria quickly.In recent years,studies have shown that it can act on all links of the food chain,as an efficient,safe,green and easy to acquire strategy to target antibiotic-resistant foodborne pathogens.Phages are one of the most abundant life forms on earth and are widely distributed in various environments.With the development of high-throughput sequencing and metagenomics,the biological information resources of lysins can be deeply explored and enriched,which lays a foundation for the systematic analysis and understanding of the"structural characteristics"-"function exertion"mode of lysins,and provides theoretical guidance for the rational search or personalized design of lysins.It is of practical significance to drive the establishment of high-throughput,high-efficiency and predictable intensive production platform and auxiliary expansion channels of differentiated design for the development of next-generation bacteria control strategies and the response to antibiotic resistance crisis.This research with the aim to control important aquatic pathogenic bacteria,Vibrio parahaemolyticus,on the basis of existing database information resources,by means of bioinformatics analysis,to build the expression vector of lysins.Then,exploring the enzymology properties and diversified applications thierof.Which provides theoretical basis for systematic exploration of lysins.The main research contents and results are as follows:1.Screening,bioinformatics analysis and expression of lysinsWith the rapid development of genomics and metagenomics based on high-throughput sequencing technology,bioinformation resources of lysins have been continuously excavated and enriched.Many of the lysins sequences or predicted reading frameworks in the database have not yet been validated,providing a reliable source for screening and recombinant expression of navol lysins.In this study,biological information resources of vibrio lysins were systematically collected and analyzed using NCBI database.Ten types of conservative functional domains were found in this study:Lyz-like superfamily,Lyz-like superfamily&PG＿binding＿1 superfamily,Lyz-like superfamily&PG＿binding＿3 superfamily,PG＿binding＿1 superfamily&NLPC＿P60superfamily,Hydrolase＿2 Superfamily,PGRP Super Family,Peptidase＿M15Superfamily,Murnac-LAA Superfamily,Zli S and Phage＿lysis Superfamily.Further analysis of the physicochemical properties and modeling analysis revealed that the common characteristics of these lysins were that the cationic residues were mostly distributed in theα-helices at the C-terminal,forming"protruding residues"to facilitate the penetration of the outer membrane barrier.Three phage lysins,Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis,were obtained by screening,optimizing and expressing through systematic bioinformatics analysis.2.Enzymatic properties of Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlisIn order to determine the enzymatic properties of Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis for theoretical guidance of their practical application,this study systematically explored the effects of temperature,pH,outer membrane penetrant,NaCl concentration and metal ions on enzyme activity.The Hydrolytic activity of peptidoglycan and safety as well as lytic activity to 63 strains of Vibrio parahaemolyticus of Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis were studied.It was found that Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis could kill more than 99%of the vibrio parahemolyticus pretreated with chloroform in 30 min,indicating that all of them had high hydrolytic activity of peptidoglycan,and 1 m M EDTA could also significantly promote the osmosis of the out membrane.Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis showed alkaline enzyme properties and maintained high activity in the range of pH=8-10,below 45℃and in 400-700 m M NaCl solution.Lyz＿V＿pgrp is more affected by metal,while Lyz＿V＿pgp60 and Lyz＿V＿zlis are relatively stable.Lyz＿V＿pgp60 and Lyz＿V＿zlis had a synergistic effect on the control of Vibrio parahaemolyticus.The combination of Lyz＿V＿zlis and Lyz＿V＿pgp60 showed varying control effects on 63vibrio parahaemolyticus strains,and the maximum bacterial load was killed by 48.56%after 30 min treatment.And Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis are safe and effective strategies for vibrio parahaemolyticus control in both cytotoxicity and in vivo experiments in mice.3.Diversified application of Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlisLyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis have high hydrolytic activity of biofilm.The degradation of vibrio parahaemolyticus biofilm preformed at 48 h is more than 90%,and the inhibition of Vibrio parahaemolyticus biofilm formation within 48 h is more than 80%.Lyz＿V＿pgrp and Lyz＿V＿zlis showed stronger biodegradation activity than Lyz＿V＿pgp60.The combination of Lyz＿V＿pgp60-Lyz＿V＿zlis-Lyz＿V＿pgrp and Lyz＿V＿pgp60-Lyz＿V＿zlis did not increase biodegradation activity of biofilm.On the contrary,it is weaker than the effect of Lyz＿V＿zlis alone.Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis could cause multidrug-resistant Vibrio parahaemolyticus to be sensitive to antibiotics to varying degrees,among which Lyz＿V＿pgp60 could sensitize all tested strains to antibiotics.Lyz＿V＿pgp60 combined with Amikacin,Gentamicin and Ciprofloxacin,Levofloxacin had a synergistic effect and increased the antibacterial effect by 13%-43.42%.The combination of Lyz＿V＿pgp60-Lyz＿V＿zlis could promote this effect more significantly,30 min of synergistic treatment resulted in an additional 17.82%-88.16%death of antibiotic-resistant Vibrio parhemolyticus,and120 min of synergistic treatment could kill 99.95%of multi-drug resistant Vibrio parhemolyticus.In the food industry,Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis effectively controlled Vibrio parahaemolyticus on salmon samples,stainless steel tool materials,bamboo and polypropylene cutting board materials,resulting in a 1±0.4 Log CFU/cm~2 reduction.And in environment,Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis can reduce 0.8-0.99 Log CFU/m L vibrio parahaemolyticus in lake water and sludge.Conclusion:In this study,Vibrio parahaemolyticus was taken as the control target,the expression vector of lysins was successfully constructed based on the existing database information resources and bioinformatics analysis combined with wet experiment from the perspective of"structural domain"-"function exertion".Three lysins,Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis were obtained,and their enzymological properties and diversified applications were investigated.Lyz＿V＿pgrp,Lyz＿V＿pgp60 and Lyz＿V＿zlis have highly biofilm degradative activity,and have synergistic effect with antibiotics,that showing good application potential of biofilm detergent and antibiotic-assisted small molecules. |
