Background: An increasing prevalence of biofilm forming strains by vancomycinresistance Staphylococcus aureus (VRSA) is one of the most important causes of antimicrobial resistance. VRSA possesses various regulatory factors to form and sustain biofilm in biotic or abiotic conditions. Among them, ureolytic activity is an important factor in the stabilization of biofilms by neutralizing the acidic environment. Various urease accessory proteins are required to activate the urease enzyme inside the biofilm. Objective: To optimize the cloning, expression and purification of urease accessory protein E from VRSA for determination of the secondary structure, and functional characterization by using Berthelot’s method. Methods: BAB58453.1 gene (which encodes possible urease accessory protein E), having 38% similarity to Bacillus pasteurii UreE protein, was cloned, expressed, and purified by single-step affinity chromatography for performing secondary structural studies using circular dichroism spectroscopy, and functional analysis using Berthelot’s and crystal violet assay. Results: Structure elucidation using NMR and circular dichroism spectroscopy techniques revealed that UreE protein has a partially foldedα-helical structure. Using Berthelot's method, it was identified that the purified UreE protein has enhanced urease enzyme activity, in comparison to the control. From the results of Berthelot’s and crystal violet assays, it was deduced that the selected gene (UreE protein) plays a key role in enhancing urease enzyme activity and contributes to biofilm stability. Conclusion: Structural studies on VRSA urease accessory proteins could aid in the identification of new drug targets or the development of effective antibiofilm strategies (in combination with other drug targets) against infections caused by biofilm-producing strains.

中文翻译：

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耐万古霉素金黄色葡萄球菌 MU50 菌株脲酶辅助蛋白 E 的结构和功能分析

背景：万古霉素耐药金黄色葡萄球菌（VRSA）形成生物膜菌株的流行率不断增加，是抗菌素耐药性的最重要原因之一。VRSA 拥有多种调节因子，可在生物或非生物条件下形成和维持生物膜。其中，尿素分解活性是通过中和酸性环境稳定生物膜的重要因素。激活生物膜内的脲酶需要各种脲酶辅助蛋白。目的：优化VRSA脲酶辅助蛋白E的克隆、表达和纯化，并采用Berthelot法测定二级结构和功能表征。方法：克隆、表达并通过单步亲和层析纯化BAB58453.1基因（编码可能的脲酶辅助蛋白E），与巴氏芽孢杆菌UreE蛋白具有38％的相似性，并使用圆二色光谱进行二级结构研究，并使用 Berthelot 和结晶紫测定法进行功能分析。结果：使用核磁共振和圆二色光谱技术的结构解析表明，UreE 蛋白具有部分折叠的 α-螺旋结构。使用Berthelot法，鉴定出纯化的UreE蛋白与对照相比具有增强的脲酶活性。根据 Berthelot 和结晶紫测定的结果，推断所选基因（UreE 蛋白）在增强脲酶活性并有助于生物膜稳定性方面发挥关键作用。结论：VRSA 脲酶辅助蛋白的结构研究有助于识别新的药物靶点或开发有效的抗生物膜策略（与其他药物靶点组合）来对抗由生物膜产生菌株引起的感染。