Enteropathogenic Escherichia coli Infection Inhibits Intestinal Ascorbic Acid Uptake via Dysregulation of Its Transporter Expression

Abstract

Background: Enteropathogenic Escherichia coli (EPEC) infection causes prolonged, watery diarrhea leading to morbidity and mortality. Although EPEC infection impacts nutrient transporter function and expression in intestinal epithelial cells, the effects of EPEC infection on intestinal absorption of ascorbic acid (AA) have not yet been investigated.

Aims: To investigate the effect of EPEC infection on intestinal AA uptake process and expression of both AA transporters.

Methods: We used two experimental models: human-derived intestinal epithelial Caco-2 cells and mice. ¹⁴C-AA uptake assay, Western blot, RT-qPCR, and promoter assay were performed.

Results: EPEC (WT) as well as ΔespF and ΔespG/G2 mutant-infected Caco-2 cells showed markedly inhibited AA uptake, while other mutants (ΔescN, ΔespA, ΔespB, and ΔespD) did not affect AA uptake. Infection also reduced protein and mRNA expression levels for both hSVCT1 and hSVCT2. EPEC-infected mice showed marked inhibitory effect on AA uptake and decreased protein and mRNA expression levels for both mSVCT1 and mSVCT2 in jejunum and colon. MicroRNA regulators of SVCT1 and SVCT2 (miR103a, miR141, and miR200a) were upregulated significantly upon EPEC infection in both Caco-2 and mouse jejunum and colon. In addition, expression of the accessory protein glyoxalate reductase/hydroxypyruvate reductase (GRHPR), which regulates SVCT1 function, was markedly decreased by EPEC infection in both models.

Conclusions: These findings suggest that EPEC infection causes inhibition in AA uptake through a multifactorial dysregulation of SVCT1 and SVCT2 expression in intestinal epithelial cells.

Keywords: EPEC; GRHPR; SVCT1; SVCT2; Vitamin C; microRNA.

Figure 1.. Effect of EPEC (WT) and mutant infection on ¹⁴C-AA uptake.

Infecting Caco-2 cells monolayers with EPEC (WT) and non-pathogenic E. coli HS4 (A) and mutants [type III secretory system protein (B), structural proteins (C), and effector proteins (D)] at 100 MOI for 60 min with additional 6 h post incubation after EPEC removal. ¹⁴C-AA uptake was subsequently performed as defined (see “Methods”). Values are mean ± SE of at least three individual uptake determinations with different passages of cells. ** P < 0.05; * P < 0.01.

Figure 2.. Effect of EPEC on hSVCT1 and hSVCT2 protein and mRNA levels.

Infecting Caco-2 cells monolayers with EPEC (WT) and HS4 (100 MOI) for 60 min with additional 6 h post incubation after EPEC removal and subjected to Western blot and RT-qPCR analyses as described in “Methods”. The hSVCT1 (A) and hSVCT2 (B) levels of expression were measured by Western blotting using protein isolated from EPEC (WT), HS4, and uninfected control Caco-2 cells. The specificity of hSVCT1 and hSVCT2 specific bands were determined by competing hSVCT1 and hSVCT2 polyclonal antibodies with synthetic antigenic peptides generated against hSVCT1 and hSVCT2 polypeptides (Ci, ii). The mRNA levels of hSVCT1 (D) and hSVCT2 (E) were determined in infected and uninfected control Caco-2 cells by RT-qPCR. Values are mean ± SE of at least triplicate determinations from different passages of cells. ** P < 0.05; * P < 0.01.

Figure 3.. Effect of acute and neonatal exposure of mice to EPEC infection on ¹⁴C-AA uptake, mSVCT1 and mSVCT2 expression levels in mouse jejunum.

¹⁴C-AA uptake was measured in mouse jejunum after acute exposure of mice with EPEC (WT) infection (see “Methods”) (A). Western blot was done using 60 μg of protein obtained from both acute and neonatal exposed jejunum mucosa in order to measure the mSVCT1 (B, G) and mSVCT2 (C, H) protein levels. The specificity of mSVCT1 and mSVCT2 specific bands were determined by competing with mSVCT1 and mSVCT2 polyclonal antibodies against synthetic antigenic peptides (Di, ii). The mSVCT1 (E, I) and mSVCT2 (F, J) mRNA expression levels in mouse jejunum were determined by RT-qPCR. Values are mean ± SE of at least triplicate determinations from different mice. ** P < 0.05; * P < 0.01.

Figure 4.. Effect of acute and neonatal exposure of mice to EPEC infection on ¹⁴C-AA uptake, mSVCT1 and mSVCT2 expression levels in mouse colon.

¹⁴C-AA uptake was determined in mouse colon after acute exposure of EPEC (WT) infection (see “Methods”) (A). Western blot was conducted using 60 μg of protein obtained from both acute and neonatal exposed colon mucosa in order to assess the mSVCT1 (B, F) and mSVCT2 (C, G) protein levels. The mSVCT1 (D, H) and mSVCT2 (E, I) mRNA expression levels in mouse colon were measured using RT-qPCR. Data are mean ± SE of at least triplicate determinations from different mice. ** P < 0.05.

Figure 5.. Effect of EPEC on SLC23A1 and SLC23A2 promoter activity.

Caco-2 cells were transiently transfected with pGL3-SLC23A1 (A) and pGL3-SLC23A2 (B) promoter constructs followed by infection with EPEC as described in “Methods”. Values are mean ± SE of at least triplicate determinations with different passages of cells.

Figure 6.. Effect of EPEC on mature microRNA levels in Caco-2 cells and mouse intestinal mucosa.

Levels of mature miR103a (A, D, G), miR141 (B, E, H), and miR200a (C, F, I) expression were determined in EPEC infected Caco-2 cells, mouse jejunum and colon as detailed in “Methods”. Data are mean ± SE of at least triplicate determinations using different passages of Caco-2 cells or tissue from multiple mice. ** P < 0.05; * P < 0.01.

Figure 7.. Effect of EPEC infection on GRHPR mRNA expression in in vitro and in vivo models.

The level of GRHPR mRNA expression in EPEC infected Caco-2 cells (A), mouse jejunum (B) and colon (C) were measured by PCR amplification. Data are mean ± SE of at least 3 individual observations from multiple samples. ** P < 0.05; * P < 0.01.