Cyclic AMP-CRP Modulates the Cell Morphology of Klebsiella pneumoniae in High-Glucose Environment

Abstract

Bacteria can modify their morphology in response to environmental stimuli for survival or host defense evasion. The rich glucose in vivo or in the Luria-Bertani (LB) medium shortened the cell length of Klebsiella pneumoniae. The environmental glucose decreased the levels of cyclic AMP (cAMP) and the transcription of crp, which declined the cAMP-cAMP receptor protein (cAMP-CRP) activity. The cell length of crp deletion mutant was significantly shorter than that of the wild type (0.981 ± 0.057 μm vs. 2.415 ± 0.075 μm, P < 0.001). These results indicated that the high environmental glucose alters the bacterial morphology to a round form through regulating the activity of cAMP-CRP complex. Comparative proteomics analysis showed increased expression of 10 proteins involved in cell division or cell wall biosynthesis in the crp deletion strain. Five of them (ompA, tolB, ybgC, ftsI, and rcsF) were selected to verify their expression in the high-glucose environment, and overexpression of tolB or rcsF shortened the bacterial length similar to that of the crp deletion strain. Electrophoretic mobility shift assay indicated that CRP directly negatively regulates the transcription of tolB and rcsF by binding to the promoter regions. This study first proved the role and partial regulation mechanism of CRP in altering cell morphology during infection and provided a theoretical basis for elucidating the mechanism in diabetes mellitus susceptible to K. pneumoniae.

Keywords: Klebsiella pneumoniae; cell morphology; cyclic AMP-CRP; glucose; regulation.

FIGURE 1

Glucose addition or crp knockout affected the morphology of Klebsiella pneumoniae NTUH-K2044. (A) The morphology of the WT strain in the infected mice liver without or with type 2 diabetes was observed after Gram-staining. Mice infects with K. pneumoniae by intraperitoneal injection. T2DM, type 2 diabetes mellitus mice, control indicates healthy mice. (B) The lengths of K. pneumoniae in (A) were analyzed by ImageJ plugin. (C) The morphology of the WT strain cultured in LB medium without or with 12 mM glucose was observed using a microscope by 1,000 × magnification. (D) The lengths of strains were analyzed by ImageJ. (E) Scanning electron micrographs of WT, Δcrp, and C-crp strains. (F) Quantitative analysis of the lengths of different strains of K. pneumoniae. WT, wild type; Δcrp, crp deletion strain; C-crp, crp complementary strain. Error bars represent standard deviation. ^∗∗P < 0.01; ^∗∗∗P < 0.001; for comparison between each group with wild type as calculated by unpaired two-tailed Student’s t-test.

FIGURE 2

Analysis of the mass spectrometry data. (A) Volcano plot from the mass spectrometry data demonstrates the magnitude and significance of the cellular proteins from the deletion strain compared with the control (WT). Horizontal dashed line shows where p-value is 0.05 [-log₁₀ (0.05) = 1.3], and the vertical dashed lines show where the fold change is 2 [log₂ (2) = 1] or 0.5 [log₂ (0.5) = -1]. The twofold change and p-value of 0.05 were used as the threshold cutoff. (B) The number of proteins was counted according to the fold change of log₂ (ratio).

FIGURE 3

Gene Ontology (GO) analysis and pathway enrichment analysis of the differential expression proteins. (A) GO annotation of the differential proteins identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS), including molecular function, biological process, and cellular component. (B) The enriched GO categories were analyzed by the REVIGO tool. The colored spots showed the regulatory proteins which appeared to be commonly involved in the major biological functions. (C) Pathway enrichment and gene quantity analysis of differentially expressed proteins.

FIGURE 4

Analysis of the indicated proteins in the cell division, transport, and metabolism. (A) Analysis of the protein–protein interaction of the identified proteins by STRING 9.0 and generated by Cytoscape 3.8. Medium confidence was set and disconnected nodes were hided. Nodes are proteins, and lines represent functional associations between proteins. Gene clusters associated with cell division (dark blue and yellow), maltose (triangle), and glycerol (diamond) metabolism involved in the network. (B) Functional clustering analysis of the differential proteins identified in the crp deletion and WT strain of K. pneumoniae.

FIGURE 5

Selected genes in the WT K. pneumoniae affected the cell morphology. (A) The messenger RNA (mRNA) levels of five selected genes and tolA, pal, and trmO were detected by quantitative PCR after the wild-type (WT) strain was cultured in the glucose-rich medium. Comparison was performed by one-way ANOVA. (B) Quantitation of the indicated mRNA levels in WT strains overexpressed with the selected proteins. WT transformed with empty vector acted as the negative control. Comparison was performed by one-way ANOVA. (C) The morphology of the overexpressed strains and WT of K. pneumoniae was scanned by a microscope after staining with crystal violet. (D) The lengths of the WT, Δcrp, and overexpression strains were analyzed using ImageJ. Comparison between each group with WT as calculated by unpaired two-tailed Student’s t-test. (E) The in vivo cAMP concentrations of WT strain cultured in LB broth with or without 12 mM glucose or 12 mM glucose +500 nM cAMP analog dibutyryl-cAMP (db-cAMP) were measured. Error bars represent standard deviation. Comparison was performed by one-way ANOVA. (F) The mRNA levels of crp, rcsF, and tolB in WT strain cultured in LB with 0 mM glucose, 12 mM glucose, and 12 mM glucose combined with 500 nM db-cAMP were quantified. Comparison was performed by one-way ANOVA. Error bars represent standard deviation. ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; n.s., not significant.

FIGURE 6

cAMP-CRP binding directly to tolB and rcsF promoter was identified by EMSA and lacZ fusion assay. (A) The tolB-pal-cpoB operon and the promoter sequence. The large arrows represent the open-reading frames, and the CRP binding site was marked as red color and underlined. (B) The rcsF-trmO operon and the promoter sequence. (C) LacZ fusion assay of the cAMP-CRP binding to the tolB and ftsI promoter regions. Recombinant pHRP309 plasmids were transferred into the WT, crp-knockout, and crp complementary mutant of K. pneumoniae. Error bars represent standard deviation. ^∗∗P < 0.01; ^∗∗∗P < 0.001; n.s., not significant; comparison was performed by one-way ANOVA. (D) cAMP-CRP binding directly to tolB and rcsF promoter regions was identified by EMSA. Ten nanogram unlabeled probe (cold probe) was added to compete with labeled probe for binding His₆-CRP protein.