Viviane C Oliveira, Nathália G Rosa-Garzon, Amanda C S D Rocha, Rachel M Monteiro, Yasmim G Gonçalves, Marcelo Kravicz, Maria A Juliano, Luiz Juliano, Priscyla D Marcato, Claudia H Silva-Lovato, Hamilton Cabral, Evandro Watanabe
{"title":"肽聚糖降解蛋白的特征:一种肽聚糖降解蛋白的特性:生化和抗菌特性、抗生素协同作用和给药系统创新。","authors":"Viviane C Oliveira, Nathália G Rosa-Garzon, Amanda C S D Rocha, Rachel M Monteiro, Yasmim G Gonçalves, Marcelo Kravicz, Maria A Juliano, Luiz Juliano, Priscyla D Marcato, Claudia H Silva-Lovato, Hamilton Cabral, Evandro Watanabe","doi":"10.1007/s12602-024-10374-5","DOIUrl":null,"url":null,"abstract":"<p><p>The global health threat posed by antibiotic resistance has led to new research involving bacteriophage-encoded enzymes. This study characterized a new peptidoglycan-degrading protein and evaluated its synergism with colistin and its antimicrobial efficacy when conjugated with polycationic-polymer nanoparticles. The gene that codes for endolysin in the vB_PaeM_USP2, a Pseudomonas aeruginosa bacteriophage, was cloned and expressed in Escherichia coli. The recombinant endolysin (rEnd2) was purified and its biochemical properties were determined using peptidoglycan substrate. The enzymatic activity was measured through peptidoglycan layer degradation and a decrease in turbidity of permeabilized Gram-negative bacteria. The antimicrobial activity of rEnd2, alone and in combination with colistin, was evaluated by checkerboard assay. The antibacterial activity of the cationic lipid oleylamine (OAM) conjugated with rEnd2 (OAM-rEnd2) was evaluated by time killing assay. The rEnd2 is structurally analogue with other endolysins and showed muramidase activity. The rEnd2 maintained higher activity between pH 6.0 to 7.5, had maximum activity at 35 °C, and was not affected by chaotropic and reducing reagents. It was sensitive to an increase in surfactant concentration, being inactivated by sodium dodecyl sulfate and cetyltrimethylammonium bromide. Ions exhibited neither a positive nor a negative effect on enzyme activity. The rEnd2 showed clear muralytic activity and decreased turbidity of permeabilized Gram-negative bacteria. However, it did not control bacterial growth despite the combination with an antibiotic and its complexation with polycation (OAM-rEnd2 nanoparticle conjugate). The rEnd2 did not show clear antimicrobial activity suggesting further optimization of conditions for its activity or engineering and modification.</p>","PeriodicalId":20506,"journal":{"name":"Probiotics and Antimicrobial Proteins","volume":" ","pages":""},"PeriodicalIF":4.4000,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Characterization of a Peptidoglycan-Degrading Protein: Biochemical and Antimicrobial Characteristics, Antibiotic Synergism, and Delivery System Innovation.\",\"authors\":\"Viviane C Oliveira, Nathália G Rosa-Garzon, Amanda C S D Rocha, Rachel M Monteiro, Yasmim G Gonçalves, Marcelo Kravicz, Maria A Juliano, Luiz Juliano, Priscyla D Marcato, Claudia H Silva-Lovato, Hamilton Cabral, Evandro Watanabe\",\"doi\":\"10.1007/s12602-024-10374-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The global health threat posed by antibiotic resistance has led to new research involving bacteriophage-encoded enzymes. 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Characterization of a Peptidoglycan-Degrading Protein: Biochemical and Antimicrobial Characteristics, Antibiotic Synergism, and Delivery System Innovation.
The global health threat posed by antibiotic resistance has led to new research involving bacteriophage-encoded enzymes. This study characterized a new peptidoglycan-degrading protein and evaluated its synergism with colistin and its antimicrobial efficacy when conjugated with polycationic-polymer nanoparticles. The gene that codes for endolysin in the vB_PaeM_USP2, a Pseudomonas aeruginosa bacteriophage, was cloned and expressed in Escherichia coli. The recombinant endolysin (rEnd2) was purified and its biochemical properties were determined using peptidoglycan substrate. The enzymatic activity was measured through peptidoglycan layer degradation and a decrease in turbidity of permeabilized Gram-negative bacteria. The antimicrobial activity of rEnd2, alone and in combination with colistin, was evaluated by checkerboard assay. The antibacterial activity of the cationic lipid oleylamine (OAM) conjugated with rEnd2 (OAM-rEnd2) was evaluated by time killing assay. The rEnd2 is structurally analogue with other endolysins and showed muramidase activity. The rEnd2 maintained higher activity between pH 6.0 to 7.5, had maximum activity at 35 °C, and was not affected by chaotropic and reducing reagents. It was sensitive to an increase in surfactant concentration, being inactivated by sodium dodecyl sulfate and cetyltrimethylammonium bromide. Ions exhibited neither a positive nor a negative effect on enzyme activity. The rEnd2 showed clear muralytic activity and decreased turbidity of permeabilized Gram-negative bacteria. However, it did not control bacterial growth despite the combination with an antibiotic and its complexation with polycation (OAM-rEnd2 nanoparticle conjugate). The rEnd2 did not show clear antimicrobial activity suggesting further optimization of conditions for its activity or engineering and modification.
期刊介绍:
Probiotics and Antimicrobial Proteins publishes reviews, original articles, letters and short notes and technical/methodological communications aimed at advancing fundamental knowledge and exploration of the applications of probiotics, natural antimicrobial proteins and their derivatives in biomedical, agricultural, veterinary, food, and cosmetic products. The Journal welcomes fundamental research articles and reports on applications of these microorganisms and substances, and encourages structural studies and studies that correlate the structure and functional properties of antimicrobial proteins.