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ESCHERICHIA COLI INFECTION CAUSES A DECREASE IN CYCLIC-AMP MEDIATED ION SECRETION IN HUMAN INTESTINAL EPITHELIAL CELLS
CL Alexander, WK MacNaughton
Inflammation Research Network, University of Calgary, Calgary, Alberta
BACKGROUND: Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) infections in humans cause severe, watery diarrhea characterized by intestinal lesions. Despite much research into E. coli virulence, the effect these pathogens have on enteric ion transport is not well characterized and underlying mechanisms have not been described.
METHODS: In order to investigate ion transport in vitro, we used the human intestinal cell line, T84. Cell monolayers were cultured on semi-permeable membrane supports and exposed to log phase EPEC or EHEC at a multiplicity of infection of 100 for 2 to 8 hours. Membrane monolayers were then mounted in Ussing chambers to measure ion transport induced by the cAMP activator, forskolin (FSK). Change in short circuit current (deltaIsc) and area under the curve of Isc (AUC), representative of the maximum rate and total ion secretion respectively, as well as the basal ion secretion, were measured. Whole cell protein expression of the Na+-K+-Cl–-cotransporter (NKCC-1) was measured by Western blot in similarly infected T84 monolayers.
RESULTS: The response to FSK was significantly (p<0.05) decreased after 4 hr exposure to either bacteria in terms of deltaIsc (control, 125.0±4.4; EPEC, 83.2±9.1; EHEC, 100.6±6.3 µA/cm2). However, the response to carbachol was not significantly altered at any time point. EPEC, but not EHEC, also increased the basal ion secretion after 4 hr exposure (control, 1.1±0.1; EPEC, 3.5±0.1; EHEC, 0.8±0.2 µA/cm2). Incubation with basolateral viable bacteria, heat-killed bacteria or sterile culture medium did not reproduce the altered secretion response, indicating that the effect is attachment-dependent and requires apical E. coli.
The secretory response to 8-Br-cAMP was significantly decreased with exposure to EPEC or EHEC (control, 125.1±2.3; EPEC, 92.4±5.8; EHEC, 105.4±7.1 µA/cm2), suggesting that the alteration is occurring downstream of cAMP-production. However, the residual FSK-induced ion transport seen with cells bathed in chloride-free buffer was not significantly decreased with exposure to the pathogens, indicating that transport of other ions is not affected by infection. Total cellular levels of NKCC-1 were shown by Western blot to not be changed with EPEC or EHEC exposure.
CONCLUSION: Pathogenic E. coli decrease cAMP-dependent ion secretion of T84 cells in an infection-dependent manner. This effect likely occurs downstream of adenylate cyclase activity and does not involve decreased NKCC-1 protein expression.