Enteropathogenic Escherichia coli inhibits intestinal vitamin B1 (thiamin) uptake: studies with human-derived intestinal epithelial Caco-2 cells

Abstract

Infection with the gram-negative enteropathogenic Escherichia coli (EPEC), a food-borne pathogen, represents a significant risk to human health. Whereas diarrhea is a major consequence of this infection, malnutrition also occurs especially in severe and prolonged cases, which may aggravate the health status of the infected hosts. Here we examined the effect of EPEC infection on the intestinal uptake of the water-soluble vitamin B1 (thiamin) using an established human intestinal epithelial Caco-2 cell model. The results showed that infecting Caco-2 cells with wild-type EPEC (but not with nonpathogenic E. coli, killed EPEC, or filtered supernatant) leads to a significant (P < 0.01) inhibition in thiamin uptake. Kinetic parameters of both the nanomolar (mediated by THTR-2) and the micromolar (mediated by THTR-1) saturable thiamin uptake processes were affected by EPEC infection. Cell surface expression of hTHTR-1 and -2 proteins, (determined by the biotinylation method) showed a significantly (P < 0.01) lower expression in EPEC-treated cells compared with controls. EPEC infection also affected the steady-state mRNA levels as well as promoter activity of the SLC19A2 and SLC19A3 genes. Infecting Caco-2 cells with EPEC mutants that harbor mutations in the escN gene (which encodes a putative ATPase for the EPEC type III secretion system, TTSS) or the espA, espB, or espD genes (which encode structural components of the TTSS) did not affect thiamin uptake. On the other hand, mutations in espF and espH genes (which encode effector proteins) exhibited partial inhibition in thiamin uptake. These results demonstrate for the first time that EPEC infection of human intestinal epithelial cells leads to inhibition in thiamin uptake via effects on physiological and molecular parameters of hTHTR-1 and -2. Furthermore, the inhibition appears to be dependent on a functional TTSS of EPEC.

Fig. 1.

Enteropathogenic Escherichia coli (EPEC) infection inhibits thiamin uptake in Caco-2 cells. Caco-2 cells at 5–6 days postplating were serum starved overnight and then infected with wild-type EPEC (EPEC-WT) or nonpathogenic E. coli (HS4) at 100 multiplicity of infection (MOI) for 1 h alone (A) or 1 h infection along with 6 h postincubation after EPEC removal (B). [³H]thiamin uptake was subsequently measured as described in methods. *P < 0.01 compared with control. When not shown, error bars are smaller than symbols.

Fig. 2.

Time course of EPEC effects on thiamin uptake. Caco-2 monolayers were infected with EPEC for 15, 30, 45, 60, 75, and 90 min, and [³H]thiamin uptake was measured. Values are means ± SE of 3–4 separate uptake determinations. When not shown, error bars are smaller than symbols.

Fig. 3.

Direct contact of live EPEC is necessary for the inhibition of thiamin uptake by Caco-2 cells. Confluent monolayers of Caco-2 were treated (for 1 h) with wild-type EPEC, boiled EPEC, or culture supernatant of EPEC, and initial rate of thiamin was then examined. Results represent means ± SE of at least 3 separate experiments. *P < 0.01 compared with control.

Fig. 4.

EPEC infection inhibits thiamin uptake system that operates at the nanomolar (A) and micromolar (B) ranges. Confluent monolayer of Caco-2 cells was infected with wild-type EPEC (100 MOI for 1 h along with 6 h postincubation) followed by determination of initial rate of thiamin uptake at nanomolar and micromolar concentrations of thiamin. Each uptake data is the result of at least 3 separate experiments and is presented as means ± SE. Kinetic parameters of the saturable processes were determined by using a computerized model of the Michaelis-Menten equation. When not shown, error bars are smaller than symbols.

Fig. 5.

Effect of EPEC infection on human thiamin transporter-1 (hTHTR-1; A) and hTHTR-2 (B) protein levels in Caco-2 cells. Confluent monolayer of Caco-2 cells was infected with wild-type EPEC (100 MOI for 1 h along with 6 h postincubation) and subjected to surface biotinylation with sulfo-NHS-biotin at 4°C. Western blot analysis was performed with surface and total intracellular fractions by use of specific anti-hTHTR-1 and anti-hTHTR-2 polyclonal antibodies. Image and data shown are representative of 3 separate sets of experiments. *P < 0.01 compared with control.

Fig. 6.

Effect of EPEC infection on hTHTR-1 (A) and hTHTR-2 (B) mRNA levels in Caco-2 cells. Real-time PCR analysis was performed with the use of mRNA from Caco-2 cells treated with EPEC-WT and nonpathogenic E. coli (100 MOI for 1 h along with 6 h postincubation) and the hTHTR-1 and hTHTR-2 specific primers as described in methods. All experiments were run on at least 3 separate occasions. Results of a representative experiment are shown. Values are means ± SE. *P < 0.01, **P < 0.05 compared with control.

Fig. 7.

Effect of EPEC infection on SLC19A2 (left) and SLC19A3 (right) promoter levels in Caco-2 cells. Cells were transfected with SLC19A2 and SLC19A3-luciferase reporter plasmid constructs and a control pGL3-basic vector by use of Lipofectamine 2000. Cells were then infected (1 h) with wild-type EPEC and nonpathogenic E. coli (100 MOI) followed 6 h postincubation. Luciferase assay was performed as described in methods. Firefly luciferase activity was normalized relative to the activity of simultaneously expressed Renilla luciferase. Values represent means ± SE of a representative experiment performed in triplicate. *P < 0.05 compared with control.

Fig. 8.

Involvement of a functional type III secretion system (TTSS) of EPEC in the inhibitory effects of thiamin uptake. Caco-2 cells were infected with EPEC or ΔescN mutant strain at 100 MOI for 1 h followed by 6 h postincubation, and then [³H]thiamin uptake was measured. Results represent means ± SE of 3 independent experiments performed in triplicate. *P < 0.05 compared with control.

Fig. 9.

Effect of EPEC type III structural proteins on thiamin uptake. Caco-2 cells were infected with EPEC or with one of the structural protein mutant strains of ΔespA, ΔespB, and ΔespD (100 MOI for 1 h along with 6 h postincubation) followed by measurement of [³H]thiamin uptake. Values are means ± SE of 3–4 separate uptake determinations. *P < 0.05 compared with control.

Fig. 10.

Effect of EPEC type III effector proteins on thiamin uptake. Caco-2 cells were infected with EPEC or with one of the effector protein mutant strains of ΔespF, ΔespG, ΔespH, and Δmap (100 MOI for 1 h followed by 6 h postincubation) followed by measurement of [³H]thiamin uptake. Values are means ± SE of 3–4 separate uptake determinations. *P < 0.01, **P < 0.05 compared with control.