LptD Depletion Disrupts Morphological Homeostasis and Upregulates Carbohydrate Metabolism in

In a previous 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 physiology and morphology. An CRISPR interference (CRISPRi) strain was constructed to allow control of 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 survival, (ii) upregulates carbohydrate metabolism, and (iii) synergizes with the antimicrobial activity of colistin.