对幽门螺杆菌对第三代和第四代氟喹诺酮类药物耐药性的新认识：基于常见 GyrA 突变的分子对接研究。

New insights into resistance of Helicobacter pylori against third- and fourth-generation fluoroquinolones: A molecular docking study of prevalent GyrA mutations.

机构信息

Department of Bioinformatics, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.

HPGC Research Group, Department of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran.

出版信息

Helicobacter. 2019 Oct;24(5):e12628. doi: 10.1111/hel.12628. Epub 2019 Jul 7.

DOI:10.1111/hel.12628

PMID:31282059

Abstract

BACKGROUND

Fluoroquinolones hinder bacterial DNA replication by inhibiting DNA gyrase. However, mutations, in the QRDR segment of its A subunit (GyrA), cause antibiotic resistance. Here, the interactions of levofloxacin (LVX), gemifloxacin (GXN), and moxifloxacin (MXN) with Helicobacter pylori GyrA, in LVX-resistant vs -sensitive strains, were studied.

METHODS

Levoflixacin-sensitive (n = 4) and -resistant (n = 9) H pylori strains, randomly selected from another antibiotic susceptibility study, underwent PCR amplification of gyrA gene, spanning the QRDR segment. The amplified gene fragments were sequenced and aligned. The homology model of H pylori GyrA was built based on that of Escherichia coli, and energy minimization was done. The interaction patterns of LVX, GXN, and MXN with GyrA were analyzed via molecular docking studies.

RESULTS

Sequence alignment of the 13 studied strains, created 5 categories of strains: (A) wild type-like (H pylori ATCC26695), (B) N87K-only, (C) D91N-only, (D) N87K + V94L, and (E) D91N + A97V mutations. The minimum inhibitory concentrations (MIC) for LVX-sensitive (category A) and -resistant (categories B-E) strains were <1 mg/L and ≥32 mg/L, respectively. The binding mode of GyrA in category A with LVX identified G35/N87/Y90/D91/V94/G114/S115/I116/D117/G118/D119, as key residues, some residing outside the QRDR segment. Category B strains lost only one interaction (G35), which led to elevated binding free energy (∆G) and full LVX resistance. Categories C-E lost more contacts, with higher ∆G and again full LVX resistance. GXN bound to GyrA of categories A and B via a different set of key residues, while MXN retained the lost contact (G35) in LVX-resistant, category B strains.

CONCLUSION

Using molecular docking tools, we identified the key residues responsible for interaction of GyrA with LVX, GXN, and MXN. In the presence of N87K-only mutation, the loss of one of these contacts (ie, G35) led to full LVX resistance. Yet, GXN and MXN overcame this mutation, by retaining all key contacts with GyrA.

摘要

背景

氟喹诺酮类药物通过抑制 DNA 回旋酶来抑制细菌 DNA 复制。然而，其 A 亚基（GyrA）QRDR 片段的突变导致了抗生素耐药性。在这里，研究了左氧氟沙星（LVX）、吉米沙星（GXN）和莫西沙星（MXN）与左氧氟沙星耐药和敏感株幽门螺杆菌 GyrA 的相互作用。

方法

随机从另一项抗生素药敏研究中选择了 4 株左氧氟沙星敏感（n=4）和 9 株左氧氟沙星耐药（n=9）的 H pylori 菌株，对 gyrA 基因进行 PCR 扩增，涵盖 QRDR 片段。扩增的基因片段进行测序和比对。基于大肠杆菌的同源模型构建了 H pylori GyrA 的同源模型，并进行了能量最小化。通过分子对接研究分析了 LVX、GXN 和 MXN 与 GyrA 的相互作用模式。

结果

对 13 株研究菌株的序列比对，创建了 5 类菌株：（A）野生型样（H pylori ATCC26695）、（B）仅 N87K、（C）仅 D91N、（D）N87K+V94L 和（E）D91N+A97V 突变。左氧氟沙星敏感（A 类）和耐药（B-E 类）菌株的最低抑菌浓度（MIC）分别为<1 mg/L 和≥32 mg/L。A 类中 GyrA 的结合模式确定了 G35/N87/Y90/D91/V94/G114/S115/I116/D117/G118/D119 为关键残基，其中一些残基位于 QRDR 片段之外。B 类菌株仅失去一个相互作用（G35），导致结合自由能（∆G）升高和完全左氧氟沙星耐药。C-E 类失去了更多的接触，具有更高的∆G，再次完全左氧氟沙星耐药。GXN 通过一组不同的关键残基与 A 和 B 类 GyrA 结合，而 MXN 在左氧氟沙星耐药、B 类菌株中保留了丢失的接触（G35）。