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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

Specific ion transport inhibitors blocked amebic cytotoxicity in UMUC3 cells.Inhibitors of ion channels hits from the RNAi screen were tested for the ability to block amebic cytotoxicity in vitro in UMUC3 cells. Cells were treated for 30 minutes at concentrations shown. We tested washout of inhibitors (+Washout +EH) relative to no washout(+Drug +EH). E. histolytica trophozoites were added at a ratio of 1:5 trophozoites to host cells. The addition of E. histolytica resulted in a 1:2 dilution of the inhibitors (concentrations prior to dilution are shown). LDH release was measured after 30 minutes. IC50 was determined by non-linear regression of the log10 of the inhibitor concentration vs. normalized cytotoxicity. IC50 determinations excluded drug concentrations that resulted in toxicity to UMUC3 cells in the absence of E. histolytica.
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f3: Specific ion transport inhibitors blocked amebic cytotoxicity in UMUC3 cells.Inhibitors of ion channels hits from the RNAi screen were tested for the ability to block amebic cytotoxicity in vitro in UMUC3 cells. Cells were treated for 30 minutes at concentrations shown. We tested washout of inhibitors (+Washout +EH) relative to no washout(+Drug +EH). E. histolytica trophozoites were added at a ratio of 1:5 trophozoites to host cells. The addition of E. histolytica resulted in a 1:2 dilution of the inhibitors (concentrations prior to dilution are shown). LDH release was measured after 30 minutes. IC50 was determined by non-linear regression of the log10 of the inhibitor concentration vs. normalized cytotoxicity. IC50 determinations excluded drug concentrations that resulted in toxicity to UMUC3 cells in the absence of E. histolytica.

Mentions: To further validate the role of ion transport in amebic cytotoxicity, we tested several known pharmacological inhibitors in UMUC3 cells. The broad-spectrum K+ channel inhibitors ibutilide (IBU) and tetraethylammonium chloride (TEA) inhibited amebic cytotoxicity to an undetectable level (Fig. 3). Ibutilide inhibits both K+ and Ca2+ channels and blocked amebic killing with an IC50 of ~5 μM, nearly 50-fold higher than the reported IC50 for specific inhibition of rapid component of the cardiac delayed rectifier potassium current K+ channels (0.01–2 μM)39404142. TEA blocks a range of K+ channels at different concentrations including: Ca2+-activated K+ channels (IC50: 150 μM), delayed-rectifier K+ channels (IC50 = 3 mM) and ATP-activated K+ channels (IC50: 15 mM)43. The IC50 of TEA for inhibition of amebic cytotoxicity was ~50 μM, consistent with specific inhibition of Ca2+-activated K+ channels. TEA and IBU were toxic to cells in the absence of ameba at high concentrations (>1 mM IBU, >2.34 mM TEA).


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)

Specific ion transport inhibitors blocked amebic cytotoxicity in UMUC3 cells.Inhibitors of ion channels hits from the RNAi screen were tested for the ability to block amebic cytotoxicity in vitro in UMUC3 cells. Cells were treated for 30 minutes at concentrations shown. We tested washout of inhibitors (+Washout +EH) relative to no washout(+Drug +EH). E. histolytica trophozoites were added at a ratio of 1:5 trophozoites to host cells. The addition of E. histolytica resulted in a 1:2 dilution of the inhibitors (concentrations prior to dilution are shown). LDH release was measured after 30 minutes. IC50 was determined by non-linear regression of the log10 of the inhibitor concentration vs. normalized cytotoxicity. IC50 determinations excluded drug concentrations that resulted in toxicity to UMUC3 cells in the absence of E. histolytica.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561901&req=5

f3: Specific ion transport inhibitors blocked amebic cytotoxicity in UMUC3 cells.Inhibitors of ion channels hits from the RNAi screen were tested for the ability to block amebic cytotoxicity in vitro in UMUC3 cells. Cells were treated for 30 minutes at concentrations shown. We tested washout of inhibitors (+Washout +EH) relative to no washout(+Drug +EH). E. histolytica trophozoites were added at a ratio of 1:5 trophozoites to host cells. The addition of E. histolytica resulted in a 1:2 dilution of the inhibitors (concentrations prior to dilution are shown). LDH release was measured after 30 minutes. IC50 was determined by non-linear regression of the log10 of the inhibitor concentration vs. normalized cytotoxicity. IC50 determinations excluded drug concentrations that resulted in toxicity to UMUC3 cells in the absence of E. histolytica.
Mentions: To further validate the role of ion transport in amebic cytotoxicity, we tested several known pharmacological inhibitors in UMUC3 cells. The broad-spectrum K+ channel inhibitors ibutilide (IBU) and tetraethylammonium chloride (TEA) inhibited amebic cytotoxicity to an undetectable level (Fig. 3). Ibutilide inhibits both K+ and Ca2+ channels and blocked amebic killing with an IC50 of ~5 μM, nearly 50-fold higher than the reported IC50 for specific inhibition of rapid component of the cardiac delayed rectifier potassium current K+ channels (0.01–2 μM)39404142. TEA blocks a range of K+ channels at different concentrations including: Ca2+-activated K+ channels (IC50: 150 μM), delayed-rectifier K+ channels (IC50 = 3 mM) and ATP-activated K+ channels (IC50: 15 mM)43. The IC50 of TEA for inhibition of amebic cytotoxicity was ~50 μM, consistent with specific inhibition of Ca2+-activated K+ channels. TEA and IBU were toxic to cells in the absence of ameba at high concentrations (>1 mM IBU, >2.34 mM TEA).

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