众所周知,外排泵是用于从细菌中去除诸如抗生素的有害物质的重要机制。鉴于许多抗生素通过在细菌内部积累而起作用,外排泵有助于阻力。外排泵灭活是对抗抗菌素耐药性的潜在策略,因为细菌无法抽出抗生素。我们最近发现,外排功能丧失的影响仅在活跃生长的细胞中明显。我们证明了鼠伤寒沙门氏菌的全球转录组在缓慢的生长过程中发生了急剧的变化,导致静止期细胞发生重塑,渗透性较低的包膜,防止抗生素进入细胞。这里,我们研究了删除鼠伤寒沙门氏菌主要外排泵的效果,AcrB,跨生长的全局基因转录。我们发现acrB敲除比野生型菌株SL1344晚进入静止期,并显示出负责厌氧能量代谢的基因表达增加和延长。我们设计了一个连接外排和膜电位的模型,由此,AcrB的失活防止质子通过内膜流入,从而防止超极化。AcrB的敲除或失活被证明增加膜电位。我们建议全局转录调节剂ArcBA感知到喹啉库的氧化还原状态(与细菌的膜电位相关)的变化,并通过关键的主调节剂RpoS协调从指数到固定相的转变,Rsd,Rmf。因此,外排泵的失活会影响沙门氏菌的基本生理学,可能对多种表型产生影响。重要意义我们首次证明了外排泵的停用会改变细菌的总体生理和代谢。不仅仅是对有毒物质的反应,外排泵似乎在维持膜电位和能量代谢中起关键作用。这一发现表明外排泵抑制或失活可能对抗生素活性具有不可预见的积极后果。鉴于稳定期细菌对抗生素吸收更具抗性,进入稳定期的后期会延长细菌对抗生素的积累。此外,膜超极化可能导致活性物质的产生增加,这些活性物质被认为对某些抗生素的活性很重要。最后,大体生理学的变化也可以解释外排突变体的毒力降低。
Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild-type strain SL1344 and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby
hyperpolarization. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella, with likely impacts on multiple phenotypes.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane
hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants.