Glucose significantly enhances enterotoxigenic Escherichia coli adherence to intestinal epithelial cells through its effects on heat-labile enterotoxin production

Abstract

The present study tested whether exposure of enterotoxigenic Escherichia coli (ETEC) to glucose at different concentrations in the media results in increased bacterial adherence to host cells through increased heat-labile enterotoxin (LT) production, thereby suggesting the effects are physiological. Porcine-origin ETEC strains grown in Casamino acid yeast extract medium containing different concentrations of glucose were washed and inoculated onto IPEC-J2 porcine intestinal epithelial cells to test for effects on adherence and host cell cAMP concentrations. Consistent with previous studies, all LT+ strains had higher ETEC adherence to IPEC-J2 cells than did LT- strains. Adherence of the LT- but not the LT+ strains was increased by pre-incubating the IPEC-J2 cells with LT and decreased by co-incubation with GM1 ganglioside in a dose-dependent manner (P<0.05). To determine whether the glucose concentration of the cell culture media has an effect on adherence, IPEC-J2 cells were inoculated with LT+ or LT- strains in cell culture media containing a final glucose concentration of 0, 0.25, 0.5, 1.0 or 2.0%, and incubated for 4 h. Only media containing 0.25% glucose resulted in increased adherence and cAMP levels, and this was limited to IPEC-J2 cells inoculated with LT+ strains. This study supports the hypothesis that glucose, at a concentration optimal for LT expression, enhances bacterial adherence through the promotion of LT production. Hence, these results establish the physiological relevance of the effects of glucose on LT production and provide a basis for how glucose intake may influence the severity of ETEC infection.

Figure 1. Effects of endogenous LT on adherence of isogenic porcine-origin ETEC strains to porcine intestinal epithelial (IPEC-J2) cells.

Strains were cultured in Casamino Acid Yeast Extract (CAYE) medium containing 0.25% glucose, final pH 8.5, conditions which supported the production and secretion of LT. IPEC-J2 cells were inoculated at a multiplicity of infection of 100∶1 in DMEM-F12 cell culture media. Adherence of F4ac⁺ ETEC strains was compared with that of non-fimbriated LT⁻ control strains G58-1 and DH5α after 1 and 2 h of incubation. eltAB and estB denote presence (+) or absence (−) of genes for LT and STb production, respectively. Means with different letter designations indicate adherence levels that are significantly different (P<0.05) within the same incubation time group.

Figure 2. Effects of exogenous LT on adherence of isogenic porcine-origin ETEC strains to porcine epithelial (IPEC-J2) cells.

A. Wild-type LT⁺ STb⁺ strain 2534-86. B. LT⁻ (ΔeltAB) strain MUN299. C. LT⁻ STb⁻ (ΔeltAB ΔestB) strain MUN300. D. Wild-type non-enterotoxigenic strain G58-1. IPEC-J2 cells were incubated 1 h prior to inoculation (Pre-LT) or co-incubated at the time of inoculation (Co-LT) with 100 ng/ml LT. Asterisk (*) denotes adherence levels of a given LT treatment were significantly different (P<0.05) from that of the non-LT-treated control (No LT).

Figure 3. Dose-dependent effect of exogenous LT on the adherence of porcine-origin ETEC strains to porcine epithelial (IPEC-J2) cells.

IPEC-J2 cells were pre-treated with 1, 10 or 100 ng/ml of exogenous LT 1 h prior to inoculation with wild-type LT⁺ STb⁺ strain 2534-86, LT⁻ STb⁻ (ΔeltAB ΔestB) MUN300, or LT⁺ complemented (ΔeltAB ΔestB/peltAB) strain MUN301. Asterisk (*) denotes a level of adherence significantly different (P<0.05) from that of the same strain without exogenous LT.

Figure 4. Inhibitory dose-response of exogenous GM1 ganglioside on the adherence of porcine-origin ETEC strains to porcine epithelial (IPEC-J2) cells.

IPEC-J2 cells were treated with 10 or 100 ng/ml of GM1 at the time of inoculation with wild-type LT⁺ STb⁺ strain 2534-86, LT⁻ STb⁻ (ΔeltAB ΔestB) MUN300, or LT⁺ complemented (ΔeltAB ΔestB/peltAB) strain MUN301. Asterisk (*) denotes a level of adherence significantly different (P<0.05) from that of the same strain without exogenous GM1.

Figure 5. Effect of glucose concentration of the media on LT production by wild-type LT⁺ STb⁺ strain 2534-86.

CAYE media with glucose concentrations of 0, 0.25, 0.5, 1 and 2% adjusted to pH 8.5 were inoculated with an overnight (18 h) culture of 2534-86 and LT concentrations in the culture supernatants were measured at 0, 2, 4 and 6 h of culture by GM1-ELISA. Separate Tukey’s tests for multiple means comparisons of LT secreted for different glucose concentrations at each time point were conducted. Different letter designations denote mean LT concentrations that are significantly different within a sampling time point (P<0.05).

Figure 6. Growth and pH of wild-type LT⁺ STb⁺ strain 2534-86 in CAYE medium containing glucose (A, B), fructose (C, D), or sucrose (E, F) at concentrations of 0, 0.25, 0.5, 1 and 2%.

CAYE media, pH 8.5, containing different carbohydrate sources were inoculated with an overnight (18 h) culture of 2534-86, and incubated at 37°C and 225 rpm for 6 h. Growth (OD₆₀₀) and pH were measured on samples collected at 0, 2, 4 and 6 h of culture.

Figure 7. Effect of sucrose (A) and fructose (B) in the culture medium on LT production and secretion by wild-type LT⁺ STb⁺ strain 2534-86.

CAYE media with fructose or sucrose at concentrations of 0, 0.25, 0.5, 1 and 2% were inoculated with an overnight (18 h) culture of 2534-86 and LT concentrations in samples of culture supernatant collected at 0, 2, 4 and 6 h of culture were determined by GM1-ELISA.

Figure 8. Effect of glucose in the IPEC-J2 cell culture media on adherence of porcine ETEC strains to porcine epithelial (IPEC-J2) cells.

IPEC-J2 cells in the presence of DMEM-F12 media containing 0, 0.25, 0.5, 1 and 2% glucose were inoculated with a 2 h culture of strain WAM2137 (spontaneous nalidixic acid-resistant mutant derivative of wild-type LT⁺ STb ⁺2534-86), MUN300 [LT⁻ STb⁻ (ΔeltAB ΔestB) derivative of WAM2317], or MUN301 (LT⁺ complemented derivative of strain MUN300) at a multiplicity of infection of 100∶1 and incubated for 4 h. Asterisk (*) denotes a level of adherence significantly different (P<0.05) from that of the same strain in IPEC-J2 cell culture media containing other glucose concentrations.

Figure 9. Effect of glucose in the IPEC-J2 cell culture media on cAMP levels of IPEC-J2 cells infected with porcine ETEC strains.

IPEC-J2 cells in the presence of DMEM-F12 media containing 0, 0.25, 0.5, 1% glucose and 0.25% glucose + glucose oxidase were inoculated with a 2 h culture of the strains at a multiplicity of infection of 100∶1 and incubated for 4 h. Strains included WAM2137 (spontaneous nalidixic acid-resistant mutant derivative of wild-type LT⁺ STb⁺2534-86), MUN300 [LT⁻ STb⁻ (ΔeltAB ΔestB) derivative of WAM2317], or MUN301 (LT⁺ complemented derivative of strain MUN300). Asterisk (*) denotes a level of adherence significantly different (P<0.05) from that of the same strain grown in other glucose concentrations.