NH125 Sensitizes Staphylococcus aureus to Cell Wall-Targeting Antibiotics through the Inhibition of the VraS Sensor Histidine Kinase.
NH125 Sensitizes Staphylococcus aureus to Cell Wall-Targeting Antibiotics through the Inhibition of the VraS Sensor Histidine Kinase.
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NH125 通过抑制 VraS 传感器组氨酸激酶使金黄色葡萄球菌对细胞壁靶向抗生素敏感。
DOI：
10.1128/spectrum.04861-22
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发表时间：
2023-06-15
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
金黄色葡萄球菌利用双组分调节系统VraSR来接收和传递环境应激信号，并且其通过上调细胞壁合成而参与细菌对几种抗生素的耐药性的发展。VraS抑制显示出延长或恢复几种临床使用的抗生素的功效。在这项工作中，我们研究了VraS胞内结构域（GST-VraS）的酶活性，以确定ATP酶反应的动力学参数，并表征NH 125在体外和微生物环境下的抑制作用。在不同GST-VraS浓度（0.95至9.49 μM）和温度（22至40°C）以及不同二价阳离子存在下测定自磷酸化反应的速率。在存在和不存在结合配偶体VraR的情况下，评估了NH 125（一种已知的激酶抑制剂）的活性和抑制作用。测定了抑制对细菌生长动力学和基因表达水平的影响。自磷酸化的GST-VraS速率随温度和VraR的加入而增加，镁是金属-ATP底物复合物的优选二价阳离子。NH 125的抑制机制本质上是非竞争性的，并且在VraR存在下减弱。在亚致死剂量的靶向细胞壁的抗生素羧苄青霉素和万古霉素的存在下添加NH 125导致金黄色葡萄球菌纽曼菌株生长的完全消除，并且在抗生素的存在下显著降低pbpB、blaZ和vraSR的基因表达水平。重要性这项工作的特点的活性和抑制VraS，这是一个关键的组氨酸激酶在细菌的双组分系统，参与金黄色葡萄球菌抗生素耐药性。结果显示温度、二价离子和VraR对ATP结合的活性和动力学参数的影响。ATP的KM值在设计筛选测定以发现具有高翻译潜力的强效和有效的VraS抑制剂中至关重要。我们报告了NH 125在体外以非竞争性方式抑制VraS的能力，并在存在和不存在细胞壁靶向抗生素的情况下研究了其对基因表达和细菌生长动力学的影响。NH 125有效地增强了抗生素对细菌生长的影响，并改变了受VraS调控的基因的表达，这些基因参与了对抗生素的耐药性。
Staphylococcus aureus utilizes the two-component regulatory system VraSR to receive and relay environmental stress signals, and it is implicated in the development of bacterial resistance to several antibiotics through the upregulation of cell wall synthesis. VraS inhibition was shown to extend or restore the efficacy of several clinically used antibiotics. In this work, we study the enzymatic activity of the VraS intracellular domain (GST-VraS) to determine the kinetic parameters of the ATPase reaction and characterize the inhibition of NH125 under in vitro and microbiological settings. The rate of the autophosphorylation reaction was determined at different GST-VraS concentrations (0.95 to 9.49 μM) and temperatures (22 to 40°C) as well as in the presence of different divalent cations. The activity and inhibition by NH125, which is a known kinase inhibitor, were assessed in the presence and absence of the binding partner, VraR. The effects of inhibition on the bacterial growth kinetics and gene expression levels were determined. The GST-VraS rate of autophosphorylation increases with temperature and with the addition of VraR, with magnesium being the preferred divalent cation for the metal-ATP substrate complex. The mechanism of inhibition of NH125 was noncompetitive in nature and was attenuated in the presence of VraR. The addition of NH125 in the presence of sublethal doses of the cell wall-targeting antibiotics carbenicillin and vancomycin led to the complete abrogation of Staphylococcus aureus Newman strain growth and significantly decreased the gene expression levels of pbpB, blaZ, and vraSR in the presence of the antibiotics. IMPORTANCE This work characterizes the activity and inhibition of VraS, which is a key histidine kinase in a bacterial two-component system that is involved in Staphylococcus aureus antibiotic resistance. The results show the effect of temperature, divalent ions, and VraR on the activity and the kinetic parameters of ATP binding. The value of the KM of ATP is vital in designing screening assays to discover potent and effective VraS inhibitors with high translational potential. We report the ability of NH125 to inhibit VraS in vitro in a noncompetitive manner and investigate its effect on gene expression and bacterial growth kinetics in the presence and absence of cell wall-targeting antibiotics. NH125 effectively potentiated the effects of the antibiotics on bacterial growth and altered the expression of the genes that are regulated by VraS and are involved in mounting a resistance to antibiotics.
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