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A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica.

Marie C, Verkerke HP, Theodorescu D, Petri WA - Sci Rep (2015)

Bottom Line: Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages.Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages.We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and International Health, University of Virginia School of Medicine, Charlottesville, Virginia USA.

ABSTRACT
The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K(+)) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K(+) channel expression. Inhibition of human K(+) channels by genetic silencing, pharmacologic inhibitors and with excess K(+) protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K(+) channel activation and K(+) efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

No MeSH data available.


Related in: MedlinePlus

K+ inhibitors blocked amebic cytotoxicity and K+ activation by E. histolytica in intestinal epithelial cells and macrophages.(a) Chemical inhibition of ion transport blocked amebic cytotoxicity. Cells were switched to the indicated concentrations of chemicals immediately prior to the addition of E. histolytica. (b) Specific K+ channel inhibitors blocked amebic cytotoxicity. Cells were treated with inhibitors for 30 minutes prior to the addition of E. histolytica. (c) K+ channel activation by E. histolytica. Fluorescence values correspond to thallium influx through open K+ channels. F values were normalized to the initial baseline value. E. histolytica (+EH) or vehicle (−EH) was added after 40 seconds. The mean of 3 biological replicates and the range is shown (+EH), −EH values are single measurements. (d) Inhibitors blocked K+ channel activation by E. histolytica. The mean of the area under the curve (AUC) for inhibitor-treated cells (KCl and ChoCl: 25 mM, 293B, CLO, PAX: 10 μ0) with (+EH) and without E. histolytica (−EH) is shown. The AUC (% of control) for each inhibitor was normalized to the media control (+EH was normalized to +EH control, −EH was normalized to −EH control). (+EH is the AUC of three biological replicates; error bars represent the range of measurements. *P < 0.001 calculated for each inhibitor relative to untreated cells by Fisher’s LSD test.
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f5: K+ inhibitors blocked amebic cytotoxicity and K+ activation by E. histolytica in intestinal epithelial cells and macrophages.(a) Chemical inhibition of ion transport blocked amebic cytotoxicity. Cells were switched to the indicated concentrations of chemicals immediately prior to the addition of E. histolytica. (b) Specific K+ channel inhibitors blocked amebic cytotoxicity. Cells were treated with inhibitors for 30 minutes prior to the addition of E. histolytica. (c) K+ channel activation by E. histolytica. Fluorescence values correspond to thallium influx through open K+ channels. F values were normalized to the initial baseline value. E. histolytica (+EH) or vehicle (−EH) was added after 40 seconds. The mean of 3 biological replicates and the range is shown (+EH), −EH values are single measurements. (d) Inhibitors blocked K+ channel activation by E. histolytica. The mean of the area under the curve (AUC) for inhibitor-treated cells (KCl and ChoCl: 25 mM, 293B, CLO, PAX: 10 μ0) with (+EH) and without E. histolytica (−EH) is shown. The AUC (% of control) for each inhibitor was normalized to the media control (+EH was normalized to +EH control, −EH was normalized to −EH control). (+EH is the AUC of three biological replicates; error bars represent the range of measurements. *P < 0.001 calculated for each inhibitor relative to untreated cells by Fisher’s LSD test.

Mentions: To further define the functional classes of ion transporters in physiologically relevant intestinal and immune cells we tested the ability of increased extracellular ions and specific pharmacological inhibitors to block amebic killing of HT-29 intestinal epithelial cells (IECs) and THP-1 macrophages. High extracellular KCl and K2SO4 blocked amebic cytotoxicity in IECs and macrophages. Extracellular KCl blocked killing with an IC50 of 7.64 mM in IECs and 11.9 mM in macrophages (Fig. 5a). K2SO4 showed similar inhibition of killing as KCl, indicating that the ionic effects are due to K+ rather than Cl− ions. NaCl blocked amebic cytotoxicity at higher concentrations (IEC IC50 = 21.8 mM , macrophage IC50 = 58.9 mM). Choline chloride was included as an osmotic control and inhibited cytotoxicity in macrophages (IC50 = 29.2 mM) and IECs (IC50 = 38.1 mM). Both NaCl and ChoCl at high concentrations can modulate K+ channels including Kcnma1, that are regulated by extracellular Na+ and Cl− ion concentrations51.


A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica.

Marie C, Verkerke HP, Theodorescu D, Petri WA - Sci Rep (2015)

K+ inhibitors blocked amebic cytotoxicity and K+ activation by E. histolytica in intestinal epithelial cells and macrophages.(a) Chemical inhibition of ion transport blocked amebic cytotoxicity. Cells were switched to the indicated concentrations of chemicals immediately prior to the addition of E. histolytica. (b) Specific K+ channel inhibitors blocked amebic cytotoxicity. Cells were treated with inhibitors for 30 minutes prior to the addition of E. histolytica. (c) K+ channel activation by E. histolytica. Fluorescence values correspond to thallium influx through open K+ channels. F values were normalized to the initial baseline value. E. histolytica (+EH) or vehicle (−EH) was added after 40 seconds. The mean of 3 biological replicates and the range is shown (+EH), −EH values are single measurements. (d) Inhibitors blocked K+ channel activation by E. histolytica. The mean of the area under the curve (AUC) for inhibitor-treated cells (KCl and ChoCl: 25 mM, 293B, CLO, PAX: 10 μ0) with (+EH) and without E. histolytica (−EH) is shown. The AUC (% of control) for each inhibitor was normalized to the media control (+EH was normalized to +EH control, −EH was normalized to −EH control). (+EH is the AUC of three biological replicates; error bars represent the range of measurements. *P < 0.001 calculated for each inhibitor relative to untreated cells by Fisher’s LSD test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4561901&req=5

f5: K+ inhibitors blocked amebic cytotoxicity and K+ activation by E. histolytica in intestinal epithelial cells and macrophages.(a) Chemical inhibition of ion transport blocked amebic cytotoxicity. Cells were switched to the indicated concentrations of chemicals immediately prior to the addition of E. histolytica. (b) Specific K+ channel inhibitors blocked amebic cytotoxicity. Cells were treated with inhibitors for 30 minutes prior to the addition of E. histolytica. (c) K+ channel activation by E. histolytica. Fluorescence values correspond to thallium influx through open K+ channels. F values were normalized to the initial baseline value. E. histolytica (+EH) or vehicle (−EH) was added after 40 seconds. The mean of 3 biological replicates and the range is shown (+EH), −EH values are single measurements. (d) Inhibitors blocked K+ channel activation by E. histolytica. The mean of the area under the curve (AUC) for inhibitor-treated cells (KCl and ChoCl: 25 mM, 293B, CLO, PAX: 10 μ0) with (+EH) and without E. histolytica (−EH) is shown. The AUC (% of control) for each inhibitor was normalized to the media control (+EH was normalized to +EH control, −EH was normalized to −EH control). (+EH is the AUC of three biological replicates; error bars represent the range of measurements. *P < 0.001 calculated for each inhibitor relative to untreated cells by Fisher’s LSD test.
Mentions: To further define the functional classes of ion transporters in physiologically relevant intestinal and immune cells we tested the ability of increased extracellular ions and specific pharmacological inhibitors to block amebic killing of HT-29 intestinal epithelial cells (IECs) and THP-1 macrophages. High extracellular KCl and K2SO4 blocked amebic cytotoxicity in IECs and macrophages. Extracellular KCl blocked killing with an IC50 of 7.64 mM in IECs and 11.9 mM in macrophages (Fig. 5a). K2SO4 showed similar inhibition of killing as KCl, indicating that the ionic effects are due to K+ rather than Cl− ions. NaCl blocked amebic cytotoxicity at higher concentrations (IEC IC50 = 21.8 mM , macrophage IC50 = 58.9 mM). Choline chloride was included as an osmotic control and inhibited cytotoxicity in macrophages (IC50 = 29.2 mM) and IECs (IC50 = 38.1 mM). Both NaCl and ChoCl at high concentrations can modulate K+ channels including Kcnma1, that are regulated by extracellular Na+ and Cl− ion concentrations51.

Bottom Line: Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages.Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages.We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and International Health, University of Virginia School of Medicine, Charlottesville, Virginia USA.

ABSTRACT
The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K(+)) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K(+) channel expression. Inhibition of human K(+) channels by genetic silencing, pharmacologic inhibitors and with excess K(+) protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K(+) channel activation and K(+) efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

No MeSH data available.


Related in: MedlinePlus