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。

IF 4.4 2区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Probiotics and Antimicrobial Proteins Pub Date : 2024-10-16 DOI:10.1007/s12602-024-10374-5
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
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引用次数: 0

摘要

抗生素耐药性对全球健康的威胁促使人们对噬菌体编码酶进行新的研究。这项研究对一种新的肽聚糖降解蛋白进行了鉴定,并评估了它与可乐定的协同作用以及与多阳离子聚合物纳米颗粒共轭后的抗菌效果。在大肠杆菌中克隆并表达了铜绿假单胞菌噬菌体 vB_PaeM_USP2 中编码内溶素的基因。纯化了重组内溶菌素(rEnd2),并使用肽聚糖底物测定了其生化性质。酶活性是通过肽聚糖层降解和渗透革兰氏阴性菌浊度降低来测定的。通过棋盘试验评估了 rEnd2 单独或与可乐定联合使用时的抗菌活性。阳离子脂质油胺(OAM)与 rEnd2 共轭(OAM-rEnd2)的抗菌活性通过时间杀伤试验进行了评估。rEnd2 在结构上与其他内溶酶类似,具有莽草酶活性。rEnd2 在 pH 值为 6.0 至 7.5 之间保持较高的活性,在 35 °C 时活性最高,并且不受混沌试剂和还原试剂的影响。它对表面活性剂浓度的增加很敏感,十二烷基硫酸钠和十六烷基三甲基溴化铵会使其失活。离子对酶活性既无积极影响,也无消极影响。rEnd2 具有明显的壁化活性,可降低渗透革兰氏阴性细菌的浑浊度。然而,尽管它与抗生素结合并与多阳离子复合物(OAM-rEnd2 纳米粒子共轭物)复配,却不能控制细菌的生长。rEnd2 没有显示出明显的抗菌活性,这表明需要进一步优化其活性条件或对其进行工程和改性。
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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.

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来源期刊
Probiotics and Antimicrobial Proteins
Probiotics and Antimicrobial Proteins BIOTECHNOLOGY & APPLIED MICROBIOLOGYMICROB-MICROBIOLOGY
CiteScore
11.30
自引率
6.10%
发文量
140
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.
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