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

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.

Keywords: Escherichia coli; LptD; carbohydrate metabolism; colanic acid; colistin.

Figure 1.

Gene silencing of LptD in E. coli using CRISPRi. (a) Schematic of the Lpt system in E. coli. LPS is extracted across the IM by LptB2FG together with MsbA and transported through the periplasm by the transenvelope bridge formed by LptA to finally be exported to the OM outer leaflet by LptDE. (b) Schematic of the Mobile-CRISPRi system. The system consists of a chromosomally integrated dCas9 and an sgRNA. The catalytically inactive dCas9 (pink) is coupled to an sgRNA (purple), which targets the complex to a specific location on the target lptD gene and acts as a physical barrier to RNA polymerase (green), sterically hindering transcription of the gene.

Figure 2.

(a) Growth curves of E. coli lptD knockdown mutant grown in LB broth with different concentrations of IPTG. Data represent the average of three biological replicates. (b) Expression of lptD over time quantified by RT-qPCR using increasing concentrations of inducer relative to the housekeeping gene gapA. Data represent the average of three biological replicates. (c) Colony-forming units at various concentrations of IPTG. Y-axis shows log-transformation of cell counts. Error bars are SDs of three technical replicates.

Figure 3.

Knockdown of lptD using CRISPRi leads to morphological changes over time. (a) Micrographs of the induced (1 mM) or uninduced (0 mM) CRISPRi strain sampled at 2, 4, 6, and 8 h. Scale bars: 200 µm. (b) TEM of uninduced control and (c) induced (1 mM) cells grown for 6 h, which show elongation and deterioration in cell shape.

Figure 4.

Expression change of biosynthesis and colanic acid-related genes. (a) Average expression changes of individual subsystems involved in biosynthesis processes are presented in large dots. Small dots represent the fold-change value of individual genes of the subsystem. (b) Enrichment scores of individual biosynthesis processes are presented as −log₁₀ of the P-value of enrichment. (c) Average expression changes of the various carbohydrate biosynthesis pathways. (d) Fold change of expression of the different genes found in the colanic acid biosynthesis pathway. (e) Average expression changes of the various carbohydrate biosynthesis pathways under LptD depletion and/or colistin exposure. (f) The colanic acid biosynthesis pathway in E. coli.

Figure 5.

Expression changes as a result of LptD depletion and/or colistin exposure. (a) Expression changes in the cell exterior pathways. The larger dots represent the average expression changes of the individual subsystems, and the smaller dots represent individual genes within the subsystem. (b) Expression changes in the flagellum-related genes. (c) Expression changes in the chemotaxis-related genes.