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 36 Cl -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 36 Cl -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.

Infectious diarrhoea is a significant contributor to morbidity and mortality worldwide. In bacterium-induced diarrhoea, rapid loss of fluids and electrolytes results from inhibition of the normal absorptive function of the intestine as well as the activation of secretory processes. Advances in the past 10 years in the fields of gastrointestinal physiology, innate immunity and enteric bacterial virulence mechanisms highlight the multifactorial nature of infectious diarrhoea. This Review explores the various mechanisms that contribute to loss of fluids and electrolytes following bacterial infections, and attempts to link these events to specific virulence factors and toxins.

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal foodborne pathogen associated with diarrhea, especially in infants and young children. Although EPEC produces characteristic attaching and effacing lesions and loss of microvilli, the pathophysiology of EPEC-associated diarrhea, particularly during early infection, remains elusive. The present studies were designed to examine the direct effects of EPEC infection on intestinal absorption via Na(+)/H(+) exchanger (NHE) isoforms. Caco-2 cells were infected with EPEC strain E2348/69 or nonpathogenic E. coli HB101 for a period of 60 to 120 min. Total NHE activity was significantly increased at 60 min, reaching approximately threefold increase after 90 min of EPEC infection. Similar findings were seen in HT-29 cells and T84 cells indicating that the response was not cell-line specific. Most surprising was the differential regulation of NHE2 and NHE3 by EPEC. Marked activation of NHE2 (300%) occurred, whereas significant inhibition ( approximately 50%) of NHE3 activity was induced. The activity of basolateral isoform NHE1 was also significantly increased in response to EPEC infection. Mutations that disrupted the type III secretion system (TTSS) ablated the effect of EPEC on the activity of both NHE2 and NHE3. These results suggest that EPEC, through a TTSS-dependent mechanism, exerts differential effects on NHE isoform activity in intestinal epithelial cells. Additionally, NHEs do not appear to play any role in EPEC-mediated inflammation, because the NHE inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride did not prevent EPEC-mediated IkappaBalpha degradation.

Diarrhea caused by enteric infections is a major factor in morbidity and mortality worldwide. An estimated 2-4 billion episodes of infectious diarrhea occur each year and are especially prevalent in infants. This review highlights the cellular and molecular mechanisms underlying diarrhea associated with the three classes of infectious agents, i.e., bacteria, viruses and parasites. Several bacterial pathogens have been chosen as model organisms, including Vibrio cholerae as a classical example of secretory diarrhea, Clostridium difficile and Shigella species as agents of inflammatory diarrhea and selected strains of pathogenic Escherichia coli (E. coli) to discuss the recent advances in alteration of epithelial ion absorption. Many of the recent studies addressing epithelial ion transport and barrier function have been carried out using viruses and parasites. Here, we focus on the rapidly developing field of viral diarrhea including rotavirus, norovirus and astrovirus infections. Finally we discuss Giardia lamblia and Entamoeba histolytica as examples of parasitic diarrhea. Parasites have a greater complexity than the other pathogens and are capable of creating molecules similar to those produced by the host, such as serotonin and PGE(2). The underlying mechanisms of infectious diarrhea discussed include alterations in ion transport and tight junctions as well as the virulence factors, which alter these processes either through direct effects or indirectly through inflammation and neurotransmitters.

SummaryEnteropathogenic Escherichia coli (EPEC) have been previously shown to alter sodium hydrogen exchanger 3 (NHE3) activity in human intestinal epithelial cells. To further characterize these observations, PS120 fibroblasts transfected with NHE3 were studied. EPEC E2348/69 infection decreased NHE3 activity in PS120 fibroblasts. The effect on NHE3 was enhanced when PS120 cells were co-transfected with the scaffolding/regulatory proteins NHERF1 or NHERF2 or EBP50 and E3KARP respectively. The decrease in NHE3 activity was dependent on an intact type III secretion system, although intimate attachment mediated by translocated intimin receptor was not required. Despite its ability to bind to NHERF proteins, the EPEC effector Map had no impact on the regulation of NHE activity. Instead, EspF was found to be responsible for decreased NHE3 activity. However, neither EspF-induced apoptosis nor the interaction of EspF with sorting nexin-9, an endocytic protein, were involved.

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+ and Cl-/HCO3- exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.

Enteropathogenic Escherichia coli (EPEC) increases sodium/hydrogen exchanger 2 (NHE2)-mediated sodium uptake by intestinal epithelial cells in a type III secretion-dependent manner. However, the mechanism(s) underlying these changes are not known. This study examines the role of a number of known secreted effector molecules and bacterial adhesins as well as the signaling pathways involved in this process. Deletion of the bacterial adhesins Tir and intimin had no effect on the increase in sodium/hydrogen exchanger (NHE) activity promoted by EPEC infection; however, there was a significant decrease upon deletion of the bundle-forming pili. Bacterial supernatant also failed to alter NHE activity, suggesting that direct interaction with bacteria is necessary. Analysis of the signal transduction cascades responsible for the increased NHE2 activity during EPEC infection showed that PLC increased Ca2+, as well as PKCalpha and PKCepsilon were involved in increasing NHE activity. The activation of PKCepsilon by EPEC has not been previously described nor has its role in regulating NHE2 activity. Because EPEC markedly increases NHE2 activity, this pathogen provides an exceptional opportunity to improve our understanding of this less-characterized NHE isoform.

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...