LptD depletion disrupts morphological homeostasis and upregulates carbohydrate metabolism in Escherichia coli.

FEMS microbes Pub Date : 2023-08-10 eCollection Date: 2023-01-01 DOI:10.1093/femsmc/xtad013
Frida Svanberg Frisinger, Bimal Jana, Juan C Ortiz-Marquez, Tim van Opijnen, Stefano Donadio, Luca Guardabassi
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Abstract

In a previous in silico study, we identified an essential outer membrane protein (LptD) as an attractive target for development of novel antibiotics. Here, we characterized the effects of LptD depletion on Escherichia coli physiology and morphology. An E. coli CRISPR interference (CRISPRi) strain was constructed to allow control of lptD expression. Induction of the CRISPRi system led to ∼440-fold reduction of gene expression. Dose-dependent growth inhibition was observed, where strong knockdown effectively inhibited initial growth but partial knockdown exhibited maximum overall killing after 24 h. LptD depletion led to morphological changes where cells exhibited long, filamentous cell shapes and cytoplasmic accumulation of lipopolysaccharide (LPS). Transcriptional profiling by RNA-Seq showed that LptD knockdown led to upregulation of carbohydrate metabolism, especially in the colanic acid biosynthesis pathway. This pathway was further overexpressed in the presence of sublethal concentrations of colistin, an antibiotic targeting LPS, indicating a specific transcriptional response to this synergistic envelope damage. Additionally, exposure to colistin during LptD depletion resulted in downregulation of pathways related to motility and chemotaxis, two important virulence traits. Altogether, these results show that LptD depletion (i) affects E. coli survival, (ii) upregulates carbohydrate metabolism, and (iii) synergizes with the antimicrobial activity of colistin.

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LptD缺失破坏大肠杆菌的形态稳态并上调碳水化合物代谢。
在之前的一项计算机研究中,我们确定了一种重要的外膜蛋白(LptD)是开发新型抗生素的一个有吸引力的靶点。在这里,我们描述了LptD缺失对大肠杆菌生理和形态的影响。构建大肠杆菌CRISPR干扰(CRISPRi)菌株以控制lptD的表达。CRISPRi系统的诱导导致基因表达减少约440倍。观察到剂量依赖性生长抑制,其中强敲除有效地抑制了初始生长,但部分敲除在24小时后表现出最大的总体杀伤。LptD缺失导致细胞形态变化,细胞表现出长的丝状细胞形状和脂多糖(LPS)的细胞质积累。RNA-Seq的转录谱显示,LptD敲低导致碳水化合物代谢上调,尤其是在可乐酸生物合成途径中。该途径在亚致死浓度的粘菌素(一种靶向LPS的抗生素)存在下进一步过表达,表明对这种协同包膜损伤的特异性转录反应。此外,在LptD耗竭期间暴露于粘菌素导致与运动性和趋化性相关的途径下调,这是两个重要的毒力特征。总之,这些结果表明,LptD缺失(i)影响大肠杆菌的存活,(ii)上调碳水化合物代谢,以及(iii)与粘菌素的抗菌活性协同作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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CiteScore
3.30
自引率
0.00%
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0
审稿时长
15 weeks
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