Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

Abstract

opathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl Ϫ /HCO 3 Ϫ exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl Ϫ /HCO 3 Ϫ exchange activity was measured as DIDS-sensitive 125 I Ϫ uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 M) increased apical Cl Ϫ /HCO 3 Ϫ exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis. DRA recycling; EPEC infection; endocytosis; exocytosis; clathrin; microtubules DOWNREGULATED IN ADENOMA (DRA), or SLC26A3, is an integral membrane transporter expressed on the apical membrane of epithelial cells, including the small intestine and colon. Mutations in the SLC26A3 gene are associated with congenital chloride diarrhea (CLD), characterized by voluminous diarrhea with high fecal chloride content (28). DRA knockout mice exhibit severe diarrhea and a phenotype similar to that of CLD patients (32). Emerging studies indicate that posttranslational modifications of DRA protein may underlie the pathophysiology of diarrheal diseases. For example, CLD-causing mutations induce diarrhea by causing loss of functional DRA protein at the plasma membrane, possibly via its misfolding and mistrafficking (25,36). Studies from our laboratory have shown that enteropathogenic Escherichia coli (EPEC) infection decreases DRA function and surface levels (7), which may underlie early diarrhea; however, the cellular recycling mechanisms of DRA under normal or diseased conditions are not well understood. Thus, investigation of the cellular pathways responsible for maintaining appropriate DRA levels on the apical membrane is important for understanding disease pathogenesis...

Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signaling axis to prevent recycling endosome maturation during infection

“…Glotfelty et al (2014) [8] recently described a similar observation, reporting the accumulation of internalized occludin within EPEC infected cells and an EspG1/G2-dependent accumulation of other tight junction proteins within the cytosol. Gill et al (2007) [9] described the EspG-dependent movement of the major apical anion exchanger DRA away from the PM to intracellular compartments during EPEC infection and showed that EspG-mediated reduction of DRA at the plasma membrane is due to a decrease in DRA exocytosis [10]. These observations support our hypothesis that EspG influences the protein composition of the host plasma membrane through modulation of recycling endosomes.…”

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

“…EPEC also modulates intestinal epithelial cell electrolyte transport, reducing the expression of sodium hydrogen exchanger 3 (NHE3), the major intestinal transporter for Na + absorption (Gawenis et al , 2002; Hecht et al , 2004; Hodges et al , 2008). Furthermore, EPEC alters the activity and surface expression of the intestinal Cl − /HCO 3 − exchanger, SLC26A3/DRA, resulting in a reduced Cl − uptake and its accumulation in the lumen, driving water loss (Gill et al , 2007; Gujral et al , 2015). EPEC effectors EspF, Map, Tir, and Intimin, act together to inactivate SGLT-1, a cotransporter responsible for 70% of the total fluid uptake by the small intestine (Dean et al , 2006; Meinild et al , 1998).…”

“…Map and NleH1 bind to Na + /H + exchanger regulatory factors I and II (NHERF1/2), and Map regulates NHERF1 activity (Simpson et al , 2006; Martinez et al , 2010). EPEC also inhibits the activity of the Cl − /HCO 3 − exchanger, SLC26A3 (Down Regulated in Adenoma, DRA), and this process is mediated by EspG1/G2 effectors (Gill et al , 2007; Gujral et al , 2015). …”

125Iodide as a surrogate tracer for epithelial chloride transport by the mouse large intestine in vitro

“…This price gap has greatly expanded, and 125 I − is now >1/500th the cost of 36 Cl − . More recently, 125 I − has been used to measure Cl − –HCO 3 − exchange activity and Cl − absorption by cultured Caco‐2 cells (Gujral et al., ; Kravtsov et al., ; Kumar et al., ) and everted gut sacs of mouse large intestine (Saksena et al., ), respectively. However, neither reported a direct comparison of I − with Cl − and, although the latter study was the first instance in which 125 I − was used to examine Cl − transport by epithelia, it presented only a single unidirectional flux.…”