Expression and characterization of pantothenate energy-coupling factor transporters as an anti-infective drug target.

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Protein Science Pub Date : 2024-11-01 DOI:10.1002/pro.5195
Atanaz Shams, Spyridon Bousis, Eleonora Diamanti, Walid A M Elgaher, Lucie Zeimetz, Jörg Haupenthal, Dirk J Slotboom, Anna K H Hirsch
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Abstract

This study investigates the potential of energy-coupling factor (ECF) transporters as promising anti-infective targets to combat antimicrobial resistance (AMR). ECF transporters, a subclass of ATP-binding cassette (ABC) transporters, facilitate the uptake of B-vitamins across bacterial membranes by utilizing ATP as an energy source. Vitamins are essential cofactors for bacterial metabolism and growth, and they can either be synthesized de novo or absorbed from the environment. These transporters are considered promising drug targets, underscoring the need for further research to harness their medicinal potential and develop selective inhibitors that block vitamin uptake in bacteria. Herein, we focused on the ECF transporter for pantothenate (vitamin B5) from Streptococcus pneumoniae and the ECF transporter for folate (vitamin B9) from Lactobacillus delbrueckii as a reference protein. We also included the energizing module for pantothenate along with both full transporter complexes. Initially, we transformed and purified the transporters, followed by an assessment of their thermal stability under various buffer composition, pH, and salt concentrations. Additionally, we monitored the melting temperature over six days to confirm their stability for further assays. We then measured the binding affinities of six ECF inhibitors using surface plasmon resonance (SPR) and evaluated their inhibitory effects through in vitro assays, including bacterial growth assay, whole-cell uptake, and transport-activity assays. After determining cytotoxicity in two human cell lines, we established an in vivo infection model using Galleria mellonella larvae to further validate our findings.

作为抗感染药物靶点的泛酸能量偶联因子转运体的表达和特征。
本研究探讨了能量偶联因子(ECF)转运体作为抗感染靶点的潜力,以对抗抗菌素耐药性(AMR)。ECF转运体是ATP结合盒(ABC)转运体的一个亚类,它利用ATP作为能量来源,促进细菌膜对B族维生素的吸收。维生素是细菌新陈代谢和生长所必需的辅助因子,它们既可以从头合成,也可以从环境中吸收。这些转运体被认为是很有前景的药物靶点,因此需要进一步研究利用它们的药用潜力,并开发能阻止细菌吸收维生素的选择性抑制剂。在这里,我们重点研究了肺炎链球菌的泛酸(维生素 B5)ECF 转运体和德尔布鲁贝克乳杆菌的叶酸(维生素 B9)ECF 转运体作为参考蛋白。我们还将泛酸盐的活力模块与这两种完整的转运体复合物结合在一起。首先,我们对转运体进行了转化和纯化,然后评估了它们在不同缓冲液成分、pH 值和盐浓度下的热稳定性。此外,我们还对其熔化温度进行了六天的监测,以确认其在进一步检测中的稳定性。然后,我们利用表面等离子体共振(SPR)测量了六种ECF抑制剂的结合亲和力,并通过体外实验(包括细菌生长实验、全细胞吸收实验和转运活性实验)评估了它们的抑制作用。在确定了两种人体细胞系的细胞毒性后,我们利用黑嘴鸥幼虫建立了一个体内感染模型,以进一步验证我们的研究结果。
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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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