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 3

The role of EPEC effector proteins.

Options: View larger image (or click on image) Download as PowerPoint
The role of EPEC effector proteins. (A) Involvement of espF, espG, espH,...
(A) Involvement of espF, espG, espH, or map in the effects of EPEC. The role of secreted effector molecules was investigated by infecting Caco-2 cells with EPEC or espF, espG, espH, and map mutants for 60 minutes. Cl–/OH– exchange activity was measured in base-loaded cells as DIDS-sensitive (300 μM) 36Cl– uptake. espF, espH, or map mutants inhibited Cl–/OH– exchange activity to the same extent as wild-type EPEC. The EspG mutant caused a relatively attenuated response as compared with wild-type EPEC. Results represent mean ± SEM of 3 separate experiments performed in triplicate. *P < 0.05 compared with control; **P < 0.05 compared with wild-type EPEC. (B) Role of espG and espG2. Caco-2 monolayers were infected with either the espG mutant, espG paralog espG2 mutant, or double mutant containing deletion of espG and espG2 for 60 minutes. Cl–/OH– exchange activity was measured in base-loaded cells as DIDS-sensitive (300 μM) 36Cl– uptake. espG and espG2 mutants significantly attenuated the inhibition in Cl– uptake compared with wild type EPEC. The double mutant completely abolished the inhibitory response. Complementation of espG in the espG mutant or in the double mutant restored the inhibition in Cl–/OH– exchange activity. Results represent mean ± SEM of 3–6 separate experiments performed in triplicate. *P < 0.05 compared with control; **P < 0.05 compared with wild-type EPEC.