基于活性的粪肠球菌丝氨酸水解酶和青霉素结合蛋白特征分析

J. S. Grunnvåg, K. Hegstad, Christian S. Lentz
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引用次数: 0

摘要

粪肠球菌是一种肠道共生细菌,作为一种机会性的医院内病原体,其重要性与日俱增。它的高水平内在和获得性抗菌药耐药性正导致治疗选择的缺乏,尤其是对万古霉素耐药菌株的感染,因此需要优先鉴定和功能验证新的药物靶点。在这里,我们利用基于活性的蛋白质分析(ABPP)--一种使用功能化共价抑制剂的化学蛋白质组学方法--检测了 11 株粪肠球菌和乳酸肠球菌的活性丝氨酸水解酶。丝氨酸水解酶是一个庞大而多样化的酶家族,其中包括青霉素结合蛋白(PBPs)等已知的药物靶标,而其他亚家族则尚未得到充分探索。使用 Bocillin-FL 进行基于凝胶的 ABPP 比较显示了 PBP 活性随菌株和生长条件的变化。使用广泛的丝氨酸水解酶反应荧光探针氟膦酸盐-TMR进行的分析表明,粪肠球菌 A1 支系菌株之间的相似性很高,但 A2 和乳杆菌菌株之间的差异较大。为了鉴定这些丝氨酸水解酶,我们使用了一种生物素化探针类似物,以便通过液相色谱-质谱法进行富集和鉴定。我们在耐万古霉素的粪肠球菌 E745 中鉴定出了 11 个基本未定性的靶标(α,β-水解酶、SGNH-水解酶、磷脂酶、酰胺酶、肽酶),这些靶标可用于药物并可在活的耐万古霉素粪肠球菌 E745 中获得,而且可能具有重要功能,有待今后的研究加以鉴定。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Activity-based protein profiling of serine hydrolases and penicillin-binding proteins in Enterococcus faecium
Enterococcus faecium is a gut commensal bacterium that is gaining increasing relevance as an opportunistic, nosocomial pathogen. Its high level of intrinsic and acquired antimicrobial resistance is causing a lack of treatment options, particularly for infections with vancomycin-resistant strains, and prioritizes the identification and functional validation of novel druggable targets. Here, we use activity-based protein profiling (ABPP), a chemoproteomics approach using functionalized covalent inhibitors, to detect active serine hydrolases across 11 E. faecium and Enterococcus lactis strains. Serine hydrolases are a big and diverse enzyme family, that includes known drug targets such as penicillin-binding proteins (PBPs), whereas other subfamilies are underexplored. Comparative gel-based ABPP using Bocillin-FL revealed strain- and growth condition dependent variations in PBP activities. Profiling with the broadly serine hydrolase-reactive fluorescent probe fluorophosphonate-TMR showed a high similarity across E. faecium clade A1 strains, but higher variation across A2 and E. lactis strains. To identify these serine hydrolases, we used a biotinylated probe analog allowing for enrichment and identification via liquid chromatography-mass spectrometry. We identified 11 largely uncharacterized targets (α,β-hydrolases, SGNH-hydrolases, phospholipases, amidases, peptidases) that are druggable and accessible in live vancomycin-resistant E. faecium E745 and may possess vital functions that are to be characterized in future studies.
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来源期刊
CiteScore
3.30
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