Mechanism underlying inhibition of intestinal apical Cl–/OH– exchange following infection with enteropathogenic E. coli
Ravinder K. Gill, … , Gail Hecht, Pradeep K. Dudeja
Ravinder K. Gill, … , Gail Hecht, Pradeep K. Dudeja
Published February 1, 2007
Citation Information: J Clin Invest. 2007;117(2):428-437. https://doi.org/10.1172/JCI29625.
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Research Article Microbiology

Mechanism underlying inhibition of intestinal apical Cl–/OH– exchange following infection with enteropathogenic E. coli

Abstract

Enteropathogenic E. coli (EPEC) is a major cause of infantile diarrhea, but the pathophysiology underlying associated diarrhea is poorly understood. We examined the role of the luminal membrane Cl–/OH– exchange process in EPEC pathogenesis using in vitro and in vivo models. Cl–/OH– exchange activity was measured as OH– gradient–driven 36Cl– uptake. EPEC infection (60 minutes–3 hours) inhibited apical Cl–/OH– exchange activity in human intestinal Caco-2 and T84 cells. This effect was dependent upon the bacterial type III secretory system (TTSS) and involved secreted effector molecules EspG and EspG2, known to disrupt the host microtubular network. The microtubule-disrupting agent colchicine (100 μM, 3 hours) also inhibited 36Cl– uptake. The plasma membrane expression of major apical anion exchanger DRA (SLC26A3) was considerably reduced in EPEC-infected cells, corresponding with decreased Cl–/OH– exchange activity. Confocal microscopic studies showed that EPEC infection caused a marked redistribution of DRA from the apical membrane to intracellular compartments. Interestingly, infection of cells with an EPEC mutant deficient in espG significantly attenuated the decrease in surface expression of DRA protein as compared with treatment with wild-type EPEC. EPEC infection in vivo (1 day) also caused marked redistribution of surface DRA protein in the mouse colon. Our data demonstrate that EspG and EspG2 play an important role in contributing to EPEC infection–associated inhibition of luminal membrane chloride transport via modulation of surface DRA expression.

Authors

Ravinder K. Gill, Alip Borthakur, Kim Hodges, Jerrold R. Turner, Daniel R. Clayburgh, Seema Saksena, Ayesha Zaheer, Krishnamurthy Ramaswamy, Gail Hecht, Pradeep K. Dudeja

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Figure 4

Cell-surface expression of DRA.

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Cell-surface expression of DRA. Caco-2 monolayers grown on plastic suppo...
Caco-2 monolayers grown on plastic supports were infected with nonpathogenic E. coli, EPEC, or espG mutant for 60 minutes and subjected to biotinylation at 4°C using sulfo-NHS-biotin. After solubilization, biotinylated proteins were extracted with streptavidin-agarose from equal amounts of total cellular protein. Surface and intracellular fractions were run on 10% SDS–polyacrylamide electrophoresis gels, followed by transfer to nitrocellulose membrane. The blot was immunostained with an avidin-peroxidase antibody (A) or rabbit anti-DRA (B). Representative blot of 5 different experiments are shown. (C) Scanning densitometry of the DRA protein band was performed and the results expressed as surface/total DRA (surface plus intracellular DRA). The intensities of EPEC, nonpathogenic E. coli, and espG mutant were calculated in relation to those of the uninfected cells, and the value of each time control was arbitrarily set to 100. Values represent mean ± SEM of 5 different blots. *P < 0.01 versus control; **P < 0.05 compared with EPEC.