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Background <p>Kanamycin, an aminoglycoside, is an effective broad-spectrum antimicrobial agent against bacterial infections. However, the clinical efficacy of aminoglycosides has been overshadowed by the emergence of resistance mechanisms involving enzyme-mediated covalent modifications. Aminoglycoside nucleotidyltransferases (ANTs) inactivate aminoglycosides via AMP group transfer. Elucidating their molecular mechanisms and identifying effective inhibitors are critical for combating antimicrobial resistance.</p> Methods <p><i>Exiguobacterium </i>sp<i>.</i> PL221A was isolated from hospital sewage under selection with 16&#xa0;mg/L aminoglycosides. Following genomic sequencing using the Oxford Nanopore and BGISEQ-500 platforms, the <i>eanT</i>-1 gene encoding an ANT was identified, cloned, and expressed in <i>Escherichia coli</i> DH5α. Minimum inhibitory concentrations (MICs) against kanamycin were determined. Protein–ligand interactions between Eant-1 and kanamycin, virtual mutations of EanT-1, and inhibitor screening were assessed using the MaXFlow platform. To evaluate the catalytic mechanism, <i>eant</i>-1 mutants were expressed in vitro and their kinetic parameters were characterized. The impact of bicuculline on MICs and time-kill curves was also evaluated.</p> Results <p>Strain PL221A exhibited notable resistance to neomycin, kanamycin, and gentamicin (all MICs &gt; 128&#xa0;mg/L). Heterologous expression indicated that EanT-1 confers kanamycin resistance. Mutation of residues D43A and D98A abrogated enzymatic activity, whereas alanine substitutions at R51 and T100 had no apparent effect. Bicuculline, an isoquinoline alkaloid compound, was identified as an effective EanT-1 inhibitor, showing stronger binding affinity (˗8.72&#xa0;kcal/mol) for EanT-1 than kanamycin (˗7.51&#xa0;kcal/mol) and an ability to occupy aspartate residues critical for electrophilic polarization. Its hydroxyl-free alkaloid scaffold may prevent adenylation, enabling competitive binding with kanamycin at the catalytic site and inactivating EanT-1. Additionally, the combination of kanamycin and bicuculline effectively inhibited the growth of <i>eanT</i>-1–expressing bacteria.</p> Conclusions <p>The ANT encoded by <i>eanT</i>-1 in <i>Exiguobacterium</i> sp. PL221A mediates resistance to kanamycin, which can be counteracted by the alkaloid bicuculline through inhibition of its catalytic activity.</p>

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Bicuculline reversal of aminoglycoside O-nucleotidyltransferase EanT-1–mediated kanamycin resistance

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月3日

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  • Jiahui Wang,
  • Minghui Yu,
  • Mingzhen Zhang,
  • Zhihui Meng,
  • Peitong Yang,
  • Xingyu Cui,
  • Ziqian Yu,
  • Yongan Wang,
  • Gongli Zong

摘要

Background

Kanamycin, an aminoglycoside, is an effective broad-spectrum antimicrobial agent against bacterial infections. However, the clinical efficacy of aminoglycosides has been overshadowed by the emergence of resistance mechanisms involving enzyme-mediated covalent modifications. Aminoglycoside nucleotidyltransferases (ANTs) inactivate aminoglycosides via AMP group transfer. Elucidating their molecular mechanisms and identifying effective inhibitors are critical for combating antimicrobial resistance.

Methods

Exiguobacterium sp. PL221A was isolated from hospital sewage under selection with 16 mg/L aminoglycosides. Following genomic sequencing using the Oxford Nanopore and BGISEQ-500 platforms, the eanT-1 gene encoding an ANT was identified, cloned, and expressed in Escherichia coli DH5α. Minimum inhibitory concentrations (MICs) against kanamycin were determined. Protein–ligand interactions between Eant-1 and kanamycin, virtual mutations of EanT-1, and inhibitor screening were assessed using the MaXFlow platform. To evaluate the catalytic mechanism, eant-1 mutants were expressed in vitro and their kinetic parameters were characterized. The impact of bicuculline on MICs and time-kill curves was also evaluated.

Results

Strain PL221A exhibited notable resistance to neomycin, kanamycin, and gentamicin (all MICs > 128 mg/L). Heterologous expression indicated that EanT-1 confers kanamycin resistance. Mutation of residues D43A and D98A abrogated enzymatic activity, whereas alanine substitutions at R51 and T100 had no apparent effect. Bicuculline, an isoquinoline alkaloid compound, was identified as an effective EanT-1 inhibitor, showing stronger binding affinity (˗8.72 kcal/mol) for EanT-1 than kanamycin (˗7.51 kcal/mol) and an ability to occupy aspartate residues critical for electrophilic polarization. Its hydroxyl-free alkaloid scaffold may prevent adenylation, enabling competitive binding with kanamycin at the catalytic site and inactivating EanT-1. Additionally, the combination of kanamycin and bicuculline effectively inhibited the growth of eanT-1–expressing bacteria.

Conclusions

The ANT encoded by eanT-1 in Exiguobacterium sp. PL221A mediates resistance to kanamycin, which can be counteracted by the alkaloid bicuculline through inhibition of its catalytic activity.