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.
View: Text | PDF
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

×

Figure 6

EPEC infection causes a reduction in the expression of DRA on the plasma membrane.

Options: View larger image (or click on image) Download as PowerPoint
EPEC infection causes a reduction in the expression of DRA on the plasma...
(A) Confocal microscopic localization of DRA (red) and Alexa Fluor 488–conjugated phalloidin (actin; green) showing vertical (xy; near the apical plasma membrane) and horizontal (xz) sections. Hoechst dye labeled the nuclei (blue). At the basal level, DRA protein (red) was primarily localized in the apical membrane, with no staining in the basolateral membranes in control monolayers. EPEC-infected monolayers showed absence of DRA from the apical membrane, with marked redistribution to intracellular compartments, whereas double mutant espG/espG2 showed predominant localization of DRA on the apical membrane. Nuclear staining with Hoechst dye also shows the bacterial attachment sites as indicated by the diffused pattern of nuclei (blue) in EPEC- and double mutant–infected monolayers compared with the uniform staining pattern of the nuclei in uninfected monolayers. Scale bars: 10 μm (control and EPEC), 5 μm (ΔespG/espG2). (B) Caco-2 monolayers infected with nonpathogenic E. coli (A) or EPEC (B) were stained with DRA and phalloidin, and images were converted on 3D projections generated using AutoVisualize. In monolayers infected with nonpathogenic E. coli, DRA can be seen at the apical border associated with brush border F-actin and apical plasma membrane (arrow). As described in previous studies, EPEC infection causes a dramatic reorganization of F-actin such that it is concentrated beneath bacterial attachment sites. While some apical plasma membrane DRA could still be detected (arrow), the majority of the DRA was found in the basal cytoplasm (seen as purple due to overlap with blue nuclear stain).