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Inhibition and Redistribution of NHE3, the Apical Na+/H+ Exchanger, by Clostridium difficile Toxin B

Hayashi H, Szászi K, Coady-Osberg N, Furuya W, Bretscher AP, Orlowski J, Grinstein S - J. Gen. Physiol. (2004)

Bottom Line: We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin.Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface.The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.

Affiliation: Cell Biology Program, Hospital for Sick Children, Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada.

ABSTRACT

NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.

Effect of C. difficile Toxin B (TxB) on actin structure and NHE activity. (A–D) OK cells grown on coverslips were left untreated (A and B) or were treated with 1 μg/ml TxB for 4–6 h (C and D) before fixation. The cells were permeabilized and F-actin visualized using Alexa 488–labeled phalloidin. (A and C) Basal focal plane. (B and D) Apical focal plane. Bar, 10 μm. (E–G) Measurements of intracellular pH (pHi). OK (E), LLC-PK1 (F), and BeWo (G) cells grown on coverslips were loaded with BCECF and NH4Cl as described under materials and methods. Where indicated by the open arrow, acid loading was accomplished by removal of extracellular NH4 using a Na+-free solution. Physiological Na+-containing solution was reintroduced where noted by the black arrows. In F, 100 μM amiloride was present throughout, but was removed from the lower trace where noted by the arrowhead. Where specified, the cells were pretreated with TxB for 4–6 h. pHi was monitored fluorimetrically and calibrated as detailed in materials and methods. (H) Quantification of the rate of NHE3 (left ordinate) and NHE1 (right ordinate) activity. Data for the indicated control or TxB-treated cell types was obtained from experiments like those in E–G. Data are means ± SE of at least four experiments of each kind.
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fig1: Effect of C. difficile Toxin B (TxB) on actin structure and NHE activity. (A–D) OK cells grown on coverslips were left untreated (A and B) or were treated with 1 μg/ml TxB for 4–6 h (C and D) before fixation. The cells were permeabilized and F-actin visualized using Alexa 488–labeled phalloidin. (A and C) Basal focal plane. (B and D) Apical focal plane. Bar, 10 μm. (E–G) Measurements of intracellular pH (pHi). OK (E), LLC-PK1 (F), and BeWo (G) cells grown on coverslips were loaded with BCECF and NH4Cl as described under materials and methods. Where indicated by the open arrow, acid loading was accomplished by removal of extracellular NH4 using a Na+-free solution. Physiological Na+-containing solution was reintroduced where noted by the black arrows. In F, 100 μM amiloride was present throughout, but was removed from the lower trace where noted by the arrowhead. Where specified, the cells were pretreated with TxB for 4–6 h. pHi was monitored fluorimetrically and calibrated as detailed in materials and methods. (H) Quantification of the rate of NHE3 (left ordinate) and NHE1 (right ordinate) activity. Data for the indicated control or TxB-treated cell types was obtained from experiments like those in E–G. Data are means ± SE of at least four experiments of each kind.

Mentions: To assess the effect of TxB on NHE3, we initially selected the OK epithelial cell line. Unlike most other epithelial lines available, OK cells exclusively express NHE3 (Helmle-Kolb et al., 1990, 1993), greatly simplifying the analysis and interpretation of the data. It is noteworthy that NHE3 distribution and function is thought to be identical in renal and intestinal epithelial cells (Yun et al., 1995). Despite its comparatively large size (270 kD), TxB is capable of entering most mammalian cells, albeit slowly. Fig. 1 illustrates that OK cells are similarly susceptible to the toxin, which altered their F-actin staining pattern. Note that the well-defined stress fibers normally observed at the base of untreated OK cells (Fig. 1 A) are largely eliminated by treatment with TxB (1 μg/ml for 4–6 h; Fig. 1 C). The submembranous actin that lines the junctions between normal cells (Fig. 1 B) is also disrupted by TxB, though the apical microvilli are partially preserved (Fig. 1 D). These changes are similar to those exerted by the toxin in a variety of intestinal and renal cell lines (Hecht et al., 1992; Klussmann et al., 2001).

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Inhibition and Redistribution of NHE3, the Apical Na+/H+ Exchanger, by Clostridium difficile Toxin B

Hayashi H, Szászi K, Coady-Osberg N, Furuya W, Bretscher AP, Orlowski J, Grinstein S - J. Gen. Physiol. (2004)

Effect of C. difficile Toxin B (TxB) on actin structure and NHE activity. (A–D) OK cells grown on coverslips were left untreated (A and B) or were treated with 1 μg/ml TxB for 4–6 h (C and D) before fixation. The cells were permeabilized and F-actin visualized using Alexa 488–labeled phalloidin. (A and C) Basal focal plane. (B and D) Apical focal plane. Bar, 10 μm. (E–G) Measurements of intracellular pH (pHi). OK (E), LLC-PK1 (F), and BeWo (G) cells grown on coverslips were loaded with BCECF and NH4Cl as described under materials and methods. Where indicated by the open arrow, acid loading was accomplished by removal of extracellular NH4 using a Na+-free solution. Physiological Na+-containing solution was reintroduced where noted by the black arrows. In F, 100 μM amiloride was present throughout, but was removed from the lower trace where noted by the arrowhead. Where specified, the cells were pretreated with TxB for 4–6 h. pHi was monitored fluorimetrically and calibrated as detailed in materials and methods. (H) Quantification of the rate of NHE3 (left ordinate) and NHE1 (right ordinate) activity. Data for the indicated control or TxB-treated cell types was obtained from experiments like those in E–G. Data are means ± SE of at least four experiments of each kind.
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fig1: Effect of C. difficile Toxin B (TxB) on actin structure and NHE activity. (A–D) OK cells grown on coverslips were left untreated (A and B) or were treated with 1 μg/ml TxB for 4–6 h (C and D) before fixation. The cells were permeabilized and F-actin visualized using Alexa 488–labeled phalloidin. (A and C) Basal focal plane. (B and D) Apical focal plane. Bar, 10 μm. (E–G) Measurements of intracellular pH (pHi). OK (E), LLC-PK1 (F), and BeWo (G) cells grown on coverslips were loaded with BCECF and NH4Cl as described under materials and methods. Where indicated by the open arrow, acid loading was accomplished by removal of extracellular NH4 using a Na+-free solution. Physiological Na+-containing solution was reintroduced where noted by the black arrows. In F, 100 μM amiloride was present throughout, but was removed from the lower trace where noted by the arrowhead. Where specified, the cells were pretreated with TxB for 4–6 h. pHi was monitored fluorimetrically and calibrated as detailed in materials and methods. (H) Quantification of the rate of NHE3 (left ordinate) and NHE1 (right ordinate) activity. Data for the indicated control or TxB-treated cell types was obtained from experiments like those in E–G. Data are means ± SE of at least four experiments of each kind.
Mentions: To assess the effect of TxB on NHE3, we initially selected the OK epithelial cell line. Unlike most other epithelial lines available, OK cells exclusively express NHE3 (Helmle-Kolb et al., 1990, 1993), greatly simplifying the analysis and interpretation of the data. It is noteworthy that NHE3 distribution and function is thought to be identical in renal and intestinal epithelial cells (Yun et al., 1995). Despite its comparatively large size (270 kD), TxB is capable of entering most mammalian cells, albeit slowly. Fig. 1 illustrates that OK cells are similarly susceptible to the toxin, which altered their F-actin staining pattern. Note that the well-defined stress fibers normally observed at the base of untreated OK cells (Fig. 1 A) are largely eliminated by treatment with TxB (1 μg/ml for 4–6 h; Fig. 1 C). The submembranous actin that lines the junctions between normal cells (Fig. 1 B) is also disrupted by TxB, though the apical microvilli are partially preserved (Fig. 1 D). These changes are similar to those exerted by the toxin in a variety of intestinal and renal cell lines (Hecht et al., 1992; Klussmann et al., 2001).

Bottom Line: We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin.Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface.The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.

Affiliation: Cell Biology Program, Hospital for Sick Children, Department of Biochemistry, University of Toronto, Ontario M5G 1X8, Canada.

ABSTRACT

Background: NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium. We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform. Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment. Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin. The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3. We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin. Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface. The consequent inhibition of transport is likely to contribute to the diarrheal effects of C. difficile.

View Similar Images In: Results  - Collection
View Article: Pubmed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=2234495&rFormat=json&query=null&req=5