LptD depletion disrupts morphological homeostasis and rewires carbohydrate metabolism of Escherichia coli

Proteins required for cellular growth are classified as essential and are often used as antimicrobial targets. The outer membrane (OM) is an essential component for the Gram-negative survival and lifestyle. It is therefore considered an attractive target for development of novel antibiotics with selective activity against pathogens like Escherichia coli, but with minimal impact on the beneficial Gram-positive gut microbiota. To study the potential of LptD as an antibiotic target, we constructed an E. coli CRISPRi strain targeting the essential OM protein lptD. Induction of the CRISPRi system led to growth inhibition, and up to ~440-fold reduction of gene expression. A dose-dependent behaviour in terms of survival was observed, where a strong knockdown effectively inhibited initial growth but partial knockdown exhibited maximum overall killing after 24 h. Additionally, LptD depletion led to morphological changes where cells exhibited either a short, spherical morphology or long, filamentous cell shapes and cytoplasmic accumulation of LPS together with reduced periplasms. Transcriptional profiling showed that LptD knockdown led to an upregulation in carbohydrate metabolism, especially in the colanic acid biosynthesis pathway. This was further overexpressed during LptD depletion in the presence of sublethal colistin concentrations, indicating a transcriptional response to this synergistic envelope damage. Pathways related to motility were downregulated under combination treatment, which may limit bacterial virulence. Together, these results demonstrate that LptD depletion impacts E. coli growth, viability, morphological homeostasis, and metabolism. Therefore, targeting LptD for the development of novel antimicrobials will likely induce multi-dimensional perturbation, which is ideal for efficient bacterial killing.