YciR, a Specific 3'-Phosphodiesterase, Plays a Role in the Pathogenesis of Uropathogenic Escherichia coli CFT073

Abstract

Urinary tract infections (UTIs), with the characteristics of recurrence and resistance to antibiotics due to misuse, remain a common health and economic issue for patients. Uropathogenic Escherichia coli (UPEC), which is capable of evading the immune response by forming intracellular bacterial communities (IBCs) in the cytoplasm of bladder epithelial cells (BECs) after invasion, has been shown to be the prevailing cause of UTIs. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a small molecule responsible for eliciting the innate immune response of the host only if it has not been degraded by some phosphodiesterases (PDEs), such as YciR. The relationship between YciR and c-di-GMP levels in UPEC is inconclusive. In this study, we investigated the gene expression profile of UPEC in BECs and identified yciR as an upregulated gene. Western blot revealed that YciR enhanced the virulence of UPEC by inhibiting the phosphorylation of NF-κB. The expression of yciR could be repressed by HupB in a directly binding manner. We identified YciR, a novel PDE, and defined its possible function in innate immune evasion. We also demonstrated that YciR is an HupB-dependent PDE that degrades c-di-GMP and that a low concentration of c-di-GMP might make NF-κB less phosphorylated, thereby reducing the host's pro-inflammatory response. This is the first time that YciR has been identified as a virulence factor in the pathogenesis of UPEC. These findings further increase our understanding of the pathogenesis of UPEC and provide a theoretical basis for further studies.

Keywords: cyclic dimeric guanosine monophosphate; phosphodiesterase; urinary tract infections; uropathogenic Escherichia coli; yciR.

FIGURE 1

Clusters of orthologous groups (COGs) analysis of differentially expressed genes (DEGs) in E. coli CFT073 after 8 h of invasion of the 5637 bladder cell line. Red bars indicate upregulated genes, while blue bars indicate downregulated genes.

FIGURE 2

Features of YciR. (A) Domain identification of YciR. (B) Growth curves of WT and ΔyciR. Growth curves of WT and ΔyciR strains cultured in LB broth. Growth curves of the WT and ΔyciR strains cultured in RPMI 1640. The overnight cultured strains were diluted at a ratio of 1:1,000 and then cultured under different conditions in LB broth or RPMI 1640. The spectrophotometric value was measured at OD₆₀₀ every 10 min. (C) yciR impacted the invasion of BEC infection. The 5637 bladder cell line was seeded in 24-well plates and then infected with WT and yciR mutant strains for 8 h. CFU counts of triplicate wells were representative of three independent experiments (two-tailed t-test).

FIGURE 3

yciR promotes E. coli CFT073 colonization in the bladders of Balb/c mice in acute infections. (A) Determination of the transurethral colonization ability of WT, ΔyciR, and ΔyciRpYciR strains in the bladders of Balb/c mice at 6 hpi. (B) Determination of transurethral colonization ability of WT, ΔyciR, and ΔyciRpYciR strains in the bladders of Balb/c mice at 24 hpi. The homogenates from the infected bladders of Balb/c mice with WT and yciR mutant strains were plated on the agar for CFU counts. The bars in the graph represent the mean ± SD. Each dot represents the bladder of one mouse. Three independent experiments were performed, and p-values were calculated using a two-tailed Mann–Whitney non-parametric comparison.

FIGURE 4

YciR promoted the number of IBC formations in Balb/c mouse bladders. (A) Microscopic examination of IBC formation in mice bladders infected with WT, ΔyciR, and ΔyciRpYciR strains in Balb/c mice killed at 6 hpi after transurethral catheterization. The harvested bladders were prepared as described in the Materials and Methods section for visualization by confocal laser scanning microscopy. Observation of intracellular WT, ΔyciR, and ΔyciRpYciR (red) associated with the bi-nucleus of superficial umbrella cells of the bladder (blue). Bar = 20 μm. (B) IBC enumeration in Balb/c mice bladders infected by the WT, ΔyciR, and ΔyciRpYciR strains. The number of IBCs was counted via confocal laser microscopy for the whole bladder. A single Z-section of a superficial umbrella BEC of Balb/c revealed an IBC using confocal laser microscopy. Each dot represents the IBC number in one mouse bladder. Three independent experiments were performed, and p-values were calculated using a two-tailed Mann–Whitney non-parametric comparison.

FIGURE 5

Invasion with the WT and yciR mutant strains induced a difference in the 5637 bladder cell line to increase the phosphorylation of NF-κB. The 5637 bladder cell line was infected with WT and yciR mutants for 8 h, and p-p65 was used to measure NF-κB phosphorylation. GAPDH was used as the loading control. CL refers to cell lysis for the blank control. The error bars represent the mean ± SD, and three independent experiments were performed (two-tailed t-test).

FIGURE 6

SDS-PAGE identification of DNA pulldown using the promotor region of yciR. M, Marker; lane 1, totally bacterial lysis; lane 2, potential proteins enriched by the yciR promotor. Using the abovementioned DNA pulldown technique, we used the assumed promotor of yciR as the bait to identify potential regulatory proteins.

FIGURE 7

yciR expression in different mutant strains of E. coli CFT073 and the intracellular regulation of HupB. (A) yciR expression influenced by hupB, arcA, yebK, fucR, and ygbI mutant strains. (B) Intracellular expression of hupB and yciR when the 5637 bladder cell line was infected with E. coli CFT073. Expression was normalized with the internal control gene gyrA. The error bars represent the mean ± SD, and three independent experiments were performed (two-tailed t-test).

FIGURE 8

HupB directly binds to the yciR promotor. (A) EMSAs of the yciR promotor with the purified HupB protein. (B) lacZ fragment was used as the negative control.

FIGURE 9

Graphical abstract of YciR-mediated virulence model of uropathogenic Escherichia coli CFT073.