Heat-labile enterotoxin promotes Escherichia coli adherence to intestinal epithelial cells

Abstract

Given recent evidence suggesting that the heat-labile enterotoxin (LT) provides a colonization advantage for enterotoxigenic Escherichia coli (ETEC) in vivo, we hypothesized that LT preconditions the host intestinal epithelium for ETEC adherence. To test this hypothesis, we used an in vitro model of ETEC adherence to examine the role of LT in promoting bacterium-host interactions. We present data demonstrating that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding LT-B subunit. Rp-3',5'-cyclic AMP (cAMP), an inhibitor of protein kinase A, was sufficient to abrogate LT's ability to promote subsequent bacterial adherence. Increased adherence was not due to changes in the surface expression of the host receptor for the K88ac adhesin. Evidence is also presented for a role for bacterial sensing of host-derived cAMP in promoting adherence to host cells.

FIG. 1.

LT promotes porcine ETEC adherence to IPEC-J2 cells. (A) Quantification of adherence (CFU/ml) to IPEC-J2 cells by ETEC 2534-86 strains possessing or lacking eltAB versus time of infection. (B) Quantification of ETEC 2534-86 adherence to IPEC-J2 cells (CFU/ml) after a 2-h infection in the absence (white bars) or presence of 100 ng/ml LT, either added at 1 h preinfection (gray bars) or coinfected (black bars). The inset depicts electrophoretic separation of purified LT by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 15% resolving gel.

FIG. 2.

LT promotes human ETEC adherence to Caco-2 cells. (A) Quantification of adherence (CFU/ml) to Caco-2 cells by ETEC H10407 possessing or lacking eltA after a 2-h infection in the absence or presence of 100 ng/ml LT, either added at 1 h preinfection (gray bars) or coinfected (black bars). (B) Quantification of adherence (CFU/ml) to Caco-2 cells by ETEC field isolates in the absence (white bars) or presence (gray bars) of 100 ng/ml LT.

FIG. 3.

Exogenous and endogenous LTs promote E. coli G58-1 adherence. (A) Quantification of G58-1 adherence (CFU/ml) to IPEC-J2 cells after a 4-h infection as a function of LT concentration. (B) Change in adherence (relative to the level for unmodified G58-1) as a function of endogenous expression of the indicated enterotoxin subunits or fimbrial adhesins.

FIG. 4.

Profiling of G58-1 adherence following stimulation with pharmacological agents. (A) Change in concentration of secreted cAMP ([cAMP]) following infection or intoxication with indicated bacterial strains or chemicals. (B) Change in G58-1 adherence to IPEC-J2 cells after a 4-h infection as a function of coincubation with 1.0 μg/ml OMVs purified from the indicated bacterial strains. (C) Change in G58-1 adherence to IPEC-J2 cells after a 4-h infection following 1 h of pretreatment of IPEC-J2 cells with 50 μM cAMP, 20 μM adenosine, 100 nM ATP, 1 μg/ml PT, or 1 μM guanylin. (D) Change in G58-1 adherence to IPEC-J2 cells after a 4-h infection, with cotreatment of host cells with 100 ng/ml LT, 200 μM Rp-cAMP, or LT plus Rp-cAMP.

FIG. 5.

Stimulation of G58-1 adherence with cell-free supernatants. (A) Change in G58-1 adherence to IPEC-J2 cells after a 4-h infection as a function of coincubation of naive IPEC-J2 cells with cell-free supernatants (supt) obtained after stimulation of donor cells with the indicated bacterial strains or chemicals. Where indicated, donor cells were cotreated with 100 μM DDA (supt+DDA). (B) Change in G58-1 adherence in the presence of either purified LT with or without GM1 (soluble toxin) or supernatants obtained from LT-stimulated cells with or without GM1 (supernatant). (C) Change in G58-1 (4 h) and ETEC 2534-86 (1 h) strain adherence following pretreatment (2 h) of bacterial inocula with 20 μM adenosine (open bars) or 50 μM cAMP (gray bars). The inset depicts the influence of coincubation with tetracycline on 2534-86 adherence in the presence of cAMP. (D) Differential expression of K88ac-CAT. CAT assays were performed using ETEC 2534-86, grown to an optical density at 600 nm of 0.4 to 0.6, in CFA medium supplemented where indicated with 1 mM glucose, 1 mM cAMP, or cell-free supernatants (3%, vol/vol) derived from donor cells treated with 100 ng/ml LT for 1 h or infected with wt or ΔeltAB ETEC 2534-86 for 3 h. Data are plotted as relative CAT activities versus the bacterial culture additive. (E) FaeG adhesin expression. K88ac adhesins were extracted from ETEC 2534-86 and analyzed by immunoblotting using an anti-FaeG (α-FaeG) monoclonal antibody (43).