Investigations into the Antibacterial Mechanism of Action of Viridicatumtoxins.
Investigations into the Antibacterial Mechanism of Action of Viridicatumtoxins.
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青草毒素的抗菌作用机制研究
DOI：
10.1021/acsinfecdis.0c00031
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发表时间：
2020-07-10
影响因子：
5.3
通讯作者：
Lin, Wei
中科院分区：
文献类型：
--
作者：
Li, Weijia;Li, Li;Zhang, Chao;Cai, Yuanheng;Gao, Qiyu;Wang, Fulin;Cao, Yu;Lin, Jinzhong;Li, Jie;Shang, Zhuo;Lin, Wei
关键词：
弧菌毒素是一类罕见的四环素类抗生素，能强烈抑制耐药革兰氏阳性菌。尽管有报道显示出对十一异戊二烯焦磷酸合酶（UPPS）的体外抑制活性，但葡萄球菌毒素的生物靶标和作用机制，特别是药物-靶标相互作用，在很大程度上仍然未知。在这项研究中，粪肠球菌UPPS（EfaUPPS）的结构首次确定，发现EfaUPPS不仅可以形成一个典型的功能性二聚体，但也意想不到的非典型二聚体。然后我们观察到，病毒毒素A（VirA）和B（VirB）能够与大肠杆菌的UPPS结合。faecalis、S.金黄色葡萄球菌和E.通过体外酶抑制试验、表面等离子体共振（SPR）结合分析和体内生长抑制试验证明，弧菌毒素通过与UPPS结合而发挥抗菌作用。通过分子对接研究揭示了EfaUPPS结合口袋中与VirA和VirB相互作用的关键氨基酸残基，并通过定点突变进一步验证。EfaUPPS基因的D29A、N31A和R42A单突变可明显提高其对病毒A的亲和力，而W228A单突变可显著提高其对病毒A的抗性。翻译抑制实验表明，VirA和VirB对E. coli70S核糖体。通过分子对接研究，推测葡萄球菌毒素F环与70S核糖体螺旋34之间存在空间位阻，对核糖体有弱的抑制作用。通过对葡萄球菌毒素与UPPS和70S核糖体相互作用的结构、生物化学和计算研究，不仅揭示了葡萄球菌毒素的潜在生物学作用靶点，而且为优化葡萄球菌毒素的结构、制备具有更高抗菌活性的新衍生物提供了理论基础。
Viridicatumtoxins are a rare class of tetracycline-like antibiotics that strongly inhibit drug-resistant Gram-positive bacteria. Although reported to exhibit in vitro inhibition activity to undecaprenyl pyrophosphate synthase (UPPS), an essential enzyme in bacterial cell wall synthesis, the biological targets and mechanism of action of viridicatumtoxins, especially the drug–target interactions, remain largely unknown. In this study, the structure of Enterococcus faecalis UPPS (EfaUPPS) was first determined, uncovering that EfaUPPS can form not only a typical functional dimer but also an unexpected atypical dimer. We then observed that viridicatumtoxins A (VirA) and B (VirB) are able to bind to UPPSs of E. faecalis, S. aureus, and E. coli in a direct and high-affinity manner as evidenced by in vitro enzyme inhibition assay, surface plasmon resonance (SPR) binding analysis, and in vivo growth inhibition assay, demonstrating that viridicatumtoxins exert antibacterial effects through UPPS binding. The key amino acid residues involved in the interactions with VirA and VirB in EfaUPPS binding pocket were revealed by molecular docking studies, and further validated by site-directed mutagenesis. A single mutation of EfaUPPS at D29A, N31A, and R42A can obviously increase their affinities to VirA, while a single mutation at W228A conferred significant resistance to VirA. Moreover, translation inhibition assay showed that VirA and VirB can weakly inhibit E. coli 70S ribosome. The weak inhibition of ribosome was proposed to be attributed to steric hindrance between viridicatumtoxin ring F and 70S ribosome helix 34 by molecular docking study. Our structural, biochemical, and computational investigations on the interactions of viridicatumtoxins with UPPS and 70S ribosome not only disclosed the potential biological targets of viridicatumtoxins, but also provided a theoretical basis for structural optimization to make new viridicatumtoxin derivatives with improved antimicrobial activities.
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影响因子：
4.8
作者：
Chang, SY;Ko, TP;Wang, AHJ
通讯作者：
Wang, AHJ
影响因子：
7.3
作者：
Concha, Nestor;Huang, Jianzhong;Stavenger, Robert A.
通讯作者：
Stavenger, Robert A.
影响因子：
4.9
作者：
Schedlbauer, Andreas;Kaminishi, Tatsuya;Fucini, Paola
通讯作者：
Fucini, Paola
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
3.3
作者：
Inokoshi, Junji;Nakamura, Yuichiro;Tomoda, Hiroshi
通讯作者：
Tomoda, Hiroshi