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

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. LptD depletion leads to morphological changes and upregulates carbohydrate metabolism in Escherichia coli.

在先前的一项计算机模拟研究中，我们确定了一种必需的外膜蛋白（LptD）是开发新型抗生素的一个有吸引力的靶点。在此，我们描述了LptD缺失对大肠杆菌生理和形态的影响。构建了一个大肠杆菌CRISPR干扰（CRISPRi）菌株，以实现对lptD表达的控制。CRISPRi系统的诱导导致基因表达降低约440倍。观察到剂量依赖性的生长抑制，强烈的基因敲低有效地抑制了初始生长，但部分基因敲低在24小时后表现出最大的总体杀伤效果。LptD缺失导致形态变化，细胞呈现出长丝状的细胞形状以及脂多糖（LPS）在细胞质中的积累。通过RNA - Seq进行的转录谱分析表明，LptD基因敲低导致碳水化合物代谢上调，特别是在大肠杆菌酸生物合成途径中。在亚致死浓度的粘菌素（一种针对LPS的抗生素）存在的情况下，该途径进一步过表达，这表明对这种协同的包膜损伤有特定的转录反应。此外，在LptD缺失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性。总之，这些结果表明LptD缺失（i）影响大肠杆菌的生存，（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。 LptD缺失导致大肠杆菌的形态变化并上调碳水化合物代谢。