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<p>The MacAB-TolC tripartite efflux pump is widely distributed in Gram-negative bacteria, extruding antibiotics and virulence factors that lead to multidrug resistance and pathogenicity. However, the in situ structure and assembly mechanism of MacAB-TolC remain unclear. Here, we resolve the in situ structures of the MacAB-TolC efflux pump in <i>Escherichia coli</i> by electron cryo-tomography and subtomogram averaging. In the cells without antibiotic treatment, we observe a fully assembled MacAB-TolC pump. When <i>Escherichia coli</i> cells are treated with erythromycin, in addition to the tripartite pumps, we also discover the emergence of MacA-TolC subcomplexes without MacB, indicating flexible binding of MacB in the presence of an antibiotic substrate, which is further validated by in vivo photo-crosslinking results. Together, our data suggest the in situ assembly process of MacAB-TolC starts from the formation of MacA-TolC subcomplex, and provides insights into the design of efflux pump inhibitors.</p>

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In situ structure and assembly of the ABC-type tripartite pump MacAB-TolC

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年6月3日

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  • Tong Huo,
  • Wenfang Zhang,
  • Zhili Yu,
  • Wei Zheng,
  • Yaoming Wu,
  • Qiuyu Ren,
  • Zekai Wan,
  • Junli Cao,
  • Zhao Wang,
  • Xiaodong Shi

摘要

The MacAB-TolC tripartite efflux pump is widely distributed in Gram-negative bacteria, extruding antibiotics and virulence factors that lead to multidrug resistance and pathogenicity. However, the in situ structure and assembly mechanism of MacAB-TolC remain unclear. Here, we resolve the in situ structures of the MacAB-TolC efflux pump in Escherichia coli by electron cryo-tomography and subtomogram averaging. In the cells without antibiotic treatment, we observe a fully assembled MacAB-TolC pump. When Escherichia coli cells are treated with erythromycin, in addition to the tripartite pumps, we also discover the emergence of MacA-TolC subcomplexes without MacB, indicating flexible binding of MacB in the presence of an antibiotic substrate, which is further validated by in vivo photo-crosslinking results. Together, our data suggest the in situ assembly process of MacAB-TolC starts from the formation of MacA-TolC subcomplex, and provides insights into the design of efflux pump inhibitors.