| Salmonella is a Gram-negative food-borne pathogen.It not only caused acute,chronic or latent infections of livestock and poultry,but also often caused food-borne diseases in humans by contaminating animal-derived foods.Salmonella contamination had become a huge threat to the food industry,animal husbandry and human health.The long-term use of traditional chemical preservatives as food additives had many potential safety hazards.Meanwhile,the emergence of drug-resistant Salmonella caused by the abuse of antibiotics is also an urgent problem to be solved.Therefore,it is very urgent to develop new type of safe,effective and non-drug resistant biological antibacterial agents.Phage,as a virus that can specifically target bacteria,has been often studied as a new type of antibacterial agent because of its high sterilization efficiency.However,it was later discovered that bacteriophages are prone to lysogeny and transduction,and also easily lead to the emergence of resistant strains,so their application was subject to many restrictions.Compared with bacteriophages,endolysin,a key protein for bacteriophages to lyse bacteria,is obviously safer.Endolysin is an enzyme encoded by bacteriophages at the end of infection to lyse the host bacterial cell wall,thereby releasing progeny phage particles.Endolysin lysed the bacterial cell wall quickly,had high specificity and was not easy to produce resistance,and its antibacterial spectrum was wider than that of bacteriophages.Therefore,endolysin was also called"Enzybiotic",which means enzyme antibiotic.In summary,endolysin is considered to be a potentially safe and effective biological antibacterial agent,and should be more developed and applied to the prevention and treatment of pathogenic bacteria in clinical,agriculture and food.In this study,the functional genomics analysis of a Salmonella phage fmb-p1 with broad-spectrum lytic activity obtained its endolysin Salmcide-p1 encoding gene,and then the heterologous expression of Salmcide-p1 was purified and its antibacterial activity was identified as well as enzymatic properties,and in-depth study of the antibacterial mechanism of Salmcide-p1 and the molecular mechanism of catalytic cleavage of peptidoglycan.Finally,Salmcide-p1 was applied to food preservation.This study aimed to reveal the antibacterial mechanism of the endolysin Salmcide-p1 and molecular mechanism of catalyzing the cleavage of peptidoglycan,and evaluate antibacterial effect in food preservation applications,so as to provide theoretical basis for the design,modified transformation,development and application of new biological antibacterial agents.The specific content and results were as follows:1.Functional genomics analysis of Salmonella phage fmb-p1Salmonella phage fmb-p1 was obtained by the solid growth method and then its genomic DNA was extracted.The fmb-p1 genome draft was filled in by PCR amplification of Gaps sequence sequencing,and a complete genome-wide fine map of fmb-p1 with a total length of 40,732 bp and a GC content of 49.83%was spliced.Through functional genomics methods such as gene prediction and functional annotation,it was found that the whole genome of fmb-p1 contained 55 ORFs with an average length of 656 bp.The total length of 55 ORFs accounted for 88.53%of the whole genome sequence.The high proportion indicates that the fmb-p1 genome is organized closely.Among them,46 ORFs can encode protein products,including 16 genes encoding nucleic acid metabolism and expression-related proteins,15genes encoding structural-related proteins,and 2 genes encoding cleavage-related proteins.The fmb-p1 genome did not contain gene sequences related to lysogenic transformation,and also did not contain gene sequences encoding virulence factors and t RNA,so fmb-p1 was a safer phage.Endolysin Salmcide-p1 and holin Hol-p1 genes were closely linked in the genome sequence and partially overlapped,indicating that the cleavage mechanism of fmb-p1 was Endolysin-Holin cooperative mode.Through bioinformatics analysis,it was found that Salmcide-p1 belongs to the Lysozyme-like superfamily（Gen Bank accession number:CL00222）,and its 33-141 amino acid region was a highly conserved lysozyme functional domain（EMBL-EBI|PF00959.19）,which could specifically hydrolyze theβ-1,4 linkage between N-acetylmuramic acid residues and N-acetylglucosamine residues in peptidoglycan and theβ-1,4 linkage bond between N-acetyl-D-glucosamine residues in chitosan.The Salmcide-p1 molecule contained only a spherical structure of enzymatic catalytic domain,and there was no signal peptide and transmembrane region.2.Expression and purification of endolysin Salmcide-p1 and analysis of its antibacterial activityThe use of p T7-GST-His expression vector promoted the soluble expression of recombinant Salmcide-p1 in E.coli BL21（DE3）.After further separation and purification,the activity test showed that Salmcide-p1 had a broad-spectrum antibacterial activity against Gram-negative bacteria.Including S.typhimurium CMCC 50115,S.enteritidis CMCC 50041,S.agona CICC 21586,S.anatum CICC 21498,S.Heidelberg CICC 21487,S.Miami CICC21509,S.paratyphi-B CICC 21495,S.paratyphi-C CICC 21512,S.saintpaul CICC 21486,S.choleraesuis CICC 21493 and S.dublin CICC 21497.E.coli O157:H7 CICC 21530,E.coli ATCC 35150,Vibrio parahemolyticus CICC 21528,Shigella flexneri CMCC 51571,Proteus mirabilis CMCC 49005,Yersinia pseudotuberculosis CMCC 52225,Enterobacter sakazakii CICC 21563 and Serratia marcescens CICC 10187.But Salmcide-p1 has no antibacterial activity against Staphylococcus aureus CICC 22942 and Listeria monocytogenes CICC 21662.Meanwhile,Salmcide-p1 had no hemolytic activity at an effective concentration of 112μg/m L.The optimal reaction temperature of Salmcide-p1 was4°C,and Salmcide-p1 was a weak alkaline enzyme,and its optimal reaction p H was 8.0.Salmcide-p1 not only had broad-spectrum antibacterial activity against Gram-negative bacteria,but also had good thermal stability,p H stability and sodium salt tolerance.Therefore,Salmcide-p1 was a potential candidate for the development of new antibacterial agents and an effective enzyme preparation against Gram-negative bacterial infections.3.The antibacterial mechanism of endolysin Salmcide-p1Salmonella typhimurium CMCC 50115 was used as target cells to study the antibacterial mechanism of endolysin Salmcide-p1 against Gram-negative bacteria.The cell content leakage showed that Salmcide-p1 could quickly destroy the structure of Salmonella typhimurium within 15 minutes,causing the nucleic acid,protein,K+and Ca2+to leak.The leakage of nucleic acid,protein,K+and Ca2+was positively correlated with the Salmcide-p1dosage.The results of Annexin V-FITC and PI dual-channel cell flow cytometry experiments further confirmed that Salmcide-p1 could destroy the structural integrity of Salmonella typhimurium cells,thereby inducing cell apoptosis and necrosis.Among them,the high concentration 225μg/m L Salmcide-p1 treatment could induce more early apoptotic cells than the medium concentration 112μg/m L Salmcide-p1 treatment group,but there was no significant difference in the proportion of late apoptotic and necrotic cells between the the high concentration 225μg/m L and the medium concentration 112μg/m L Salmcide-p1treatment,and their proportion of late apoptotic and necrotic cells was twice that of the low concentration 56μg/m L Salmcide-p1 treatment.Peptidoglycan gel experiment and HPLC analysis of the peptidoglycan lysate showed that Salmcide-p1 can catalyze the lysis of Salmonella typhimurium cell wall peptidoglycan,and the same concentration of Salmcide-p1 had stronger lytic activity on Salmonella typhimurium peptidoglycan than lysozyme.The peptidoglycan cleavage kinetics experiment of Salmcide-p1 showed that Salmcide-p1 could lyse most Salmonella typhimurium peptidoglycans within 1 hour and completely lysed within2 hours.In summary,Salmcide-p1 could lyse the cell wall peptidoglycan of Salmonella typhimurium,thereby dissolving the cell wall,causing damage to the cell membrane lacking cell wall protection,and causing rapid leakage of the bacterial content,leading to cell apoptosis and necrosis.Scanning electron microscopy showed that Salmcide-p1 could not penetrate the outer membrane of Salmonella typhimurium independently,and required the assistance of the outer membrane permeating agent EDTA to reach the peptidoglycan layer of the cell wall.Research on the effect of metal ions on the antibacterial activity of Salmcide-p1 found that the presence of K+could enhance the antibacterial activity of Salmcide-p1,so K+is a non-essential activation of the antibacterial effect of Salmcide-p1.Zn2+is an inhibitor of the antibacterial effect of Salmcide-p1.4.Molecular dynamics simulation analysis of the molecular mechanism of endolysin Salmcide-p1 catalyzing the lysis of cell wall peptidoglycanQuantum chemistry calculation found that theβ-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid,the linkage between N-acetylmuramic acid and oligopeptide,and the cross-linking bond between oligopeptide of the Gram-negative peptidoglycan backbone molecule PGN were the main potential site for the catalytic cleavage of endolysin.The optimal conformation of endolysin Salmcide-p1 and PGN molecule docking was used as the initial complex system to perform 20 ns all-atom molecular dynamics simulation.During the simulation process,the root mean square deviation and gyration radius of Salmcide-p1 showed that the complex system was at approximately approx.At 12 ns,the root mean square fluctuation of Salmcide-p1 indicates that the amino acids in the range of Met1-His40 and Asp85-Gly95 are more active,and they are the main amino acid residues that interact with PGN.The molecular mechanics Poisson-Boltzmann surface area（MM-PBSA）method was used to calculate the binding free energy of Salmcide-p1 and PGN in the composite system,and to analyze the hydrogen bonds and hydrophobic interactions of Salmcide-p1 and PGN in the 20 ns equilibrium state.The results showed that the amino acid residues at Gly10-His40 in theβbarrel domain of Salmcide-p1 contributed the most to the binding free energy between Salmcide-p1 and PGN and the formation of hydrogen bonds.Meanwhile,the hydrophobic effect of the hydrophobic pocket of theβ-barrel domain was important for the ability of endolysin Salmcide-p1 to bind peptidoglycan.In addition,the results of combining the activities of the Salmcide-p1 mutants showed that theβbarrel domain was the main active center of Salmcide-p1 to catalyze the cleavage of peptidoglycan.Among them,theβ-sheets（Arg21-Lys26 and Leu34-Gly37）in theβbarrel structure were an important catalytic activity center for the cleavage of peptidoglycan by Salmcide-p1.Glu18and Tyr25 were the key amino acid residues for the catalytic cleavage of peptidoglycan by Salmcide-p1.In summary,through all-atom molecular dynamics simulation,the active center and key amino acid residues of Salmcide-p1 catalyzed the cleavage of peptidoglycan in the cell wall of Gram-negative bacteria were found,and the interaction between Salmcide-p1and peptidoglycan was revealed.The mechanism provides a theoretical basis for the modification and directed evolution of the endolysin Salmcide-p1.5.The combination of endolysin Salmcide-p1 and exogenous antibacterial agents and its prevention and control of pathogenic bacteria in skim milkFirst,the fractional inhibitory concentration index（FICI）of Salmcide-p1 combined with6 food preservatives showed that the combined use of Salmcide-p1 and EDTA-2Na,glycine,sodium nitrite,potassium sorbate,sodium diacetate and calcium propionate,respectively,all showed synergistic antibacterial effect on Salmonella typhimurium CMCC 50115 and E.coli O157:H7 CICC 21530.Among which the FICI value of the combination of Salmcide-p1+EDTA-2Na and the combination of Salmcide-p1+potassium sorbate was the smallest,indicating that the synergistic effect of the two combinations was the strongest.Secondly,the combination of Salmcide-p1 with tetrabutylammonium bromide,surfactin,sodium phytate and trisodium citrate dihydrate,respectively,showed synergistic antibacterial effect,while the combination of Salmcide-p1 with sodium dodecyl sulfate showed additive antibacterial effect.Salmcide-p1 combined with cetyl trimethyl ammonium bromide showed a synergistic inhibitory effect on E.coli O157:H7,while it showed an additive inhibitory effect on Salmonella typhimurium.In addition,Salmcide-p1 could significantly increase the antibacterial activity of three different types of antimicrobial peptides OP4,Brevibacillin V and Bacillomycin D.The combination of Salmcide-p1 with antimicrobial peptides had synergistic inhibitory effect on Salmonella typhimurium and E.coli O157:H7,while there was no significant difference in the inhibitory effect of Staphylococcus aureus CICC 22942 and Listeria monocytogenes CICC 21662,which further proved that Salmcide-p1 catalyzed the lysis of Gram-negative bacteria cell wall peptidoglycan with substrate specificity.In summary,the combination strategy of Salmcide-p1 not only enabled Salmcide-p1 to be used in vitro for the prevention of Gram-negative bacteria,but also significantly reduced the dosage of other exogenous antibacterial agents.Finally,the combination of Salmcide-p1 and EDTA-2Na was applied to the prevention and control of food-borne pathogens in skim milk.The study found that the combined strategy had a significant inhibitory effect on the growth of Salmonella typhimurium CMCC 50115 and Shigella flexneri CMCC 51571 in skim milk,and reduced the number of bacteria by approximately 2 log CFU/m L,the bacterial count can be kept below the initial inoculation level by 9 days at 4°C. |
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