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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

E. histolytica induced K+ efflux in IECs.(a) Extracellular K+ ([K+ mM]E) concentrations were measured in supernatants of HT-29 cells incubated with or without E. histolytica at a ratio of 1 trophozoite to 5 host cells for 1 hour. LDH was measured concurrently in supernatants to measure cell killing and graphed as the mean of three independent experimental values and s.e.m . **P < 0.001 (+EH) vs. (−EH) by Fisher’s LSD test. (b) HT-29 cells were loaded with the K+-sensitive fluorescent dye PBFI to measure intracellular K+ concentration ([K+]I) upon interaction with E. histolytica. Cells showed a significant reduction in [K+]I after 30 minutes of contact with E. histolytica. The mean of 3 biological replicates for each experimental condition relative to the mean of untreated wild type cells is shown; error bars represent the s.e.m. (*P < 0.05; **P < 0.005; ***P < 0.001 calculated by two-tailed t test).
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f6: E. histolytica induced K+ efflux in IECs.(a) Extracellular K+ ([K+ mM]E) concentrations were measured in supernatants of HT-29 cells incubated with or without E. histolytica at a ratio of 1 trophozoite to 5 host cells for 1 hour. LDH was measured concurrently in supernatants to measure cell killing and graphed as the mean of three independent experimental values and s.e.m . **P < 0.001 (+EH) vs. (−EH) by Fisher’s LSD test. (b) HT-29 cells were loaded with the K+-sensitive fluorescent dye PBFI to measure intracellular K+ concentration ([K+]I) upon interaction with E. histolytica. Cells showed a significant reduction in [K+]I after 30 minutes of contact with E. histolytica. The mean of 3 biological replicates for each experimental condition relative to the mean of untreated wild type cells is shown; error bars represent the s.e.m. (*P < 0.05; **P < 0.005; ***P < 0.001 calculated by two-tailed t test).

Mentions: We monitored extracellular levels of K+ levels and LDH in host cells exposed to E. histolytica over an hour. We found that increased extracellular K+ preceded elevated LDH, suggesting that K+ efflux occurred prior to cell death (Fig. 6a). To further determine if E. histolytica induced efflux of intracellular K+, cells were loaded with the K+-sensitive fluorescent intravital dye PBFI. Changes in intracellular K+ concentrations in HT-29 cells during contact with E. histolytica were monitored over 30 minutes in M199 media (KCl = 5.33 mM64). Cells co-incubated with E. histolytica displayed a significant reduction in intracellular K+ after 30 minutes (Fig. 6b). The extracellular ionic concentrations of K+ in the supernatants of HT-29 cells were measured in parallel and an increase of 0.1 mM K+ in the presence of E. histolytica was detected after 30 minutes. No increase in extracellular K+ concentration was detected in the absence of E. histolytica or in E. histolytica alone (data not shown).


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)

E. histolytica induced K+ efflux in IECs.(a) Extracellular K+ ([K+ mM]E) concentrations were measured in supernatants of HT-29 cells incubated with or without E. histolytica at a ratio of 1 trophozoite to 5 host cells for 1 hour. LDH was measured concurrently in supernatants to measure cell killing and graphed as the mean of three independent experimental values and s.e.m . **P < 0.001 (+EH) vs. (−EH) by Fisher’s LSD test. (b) HT-29 cells were loaded with the K+-sensitive fluorescent dye PBFI to measure intracellular K+ concentration ([K+]I) upon interaction with E. histolytica. Cells showed a significant reduction in [K+]I after 30 minutes of contact with E. histolytica. The mean of 3 biological replicates for each experimental condition relative to the mean of untreated wild type cells is shown; error bars represent the s.e.m. (*P < 0.05; **P < 0.005; ***P < 0.001 calculated by two-tailed t test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: E. histolytica induced K+ efflux in IECs.(a) Extracellular K+ ([K+ mM]E) concentrations were measured in supernatants of HT-29 cells incubated with or without E. histolytica at a ratio of 1 trophozoite to 5 host cells for 1 hour. LDH was measured concurrently in supernatants to measure cell killing and graphed as the mean of three independent experimental values and s.e.m . **P < 0.001 (+EH) vs. (−EH) by Fisher’s LSD test. (b) HT-29 cells were loaded with the K+-sensitive fluorescent dye PBFI to measure intracellular K+ concentration ([K+]I) upon interaction with E. histolytica. Cells showed a significant reduction in [K+]I after 30 minutes of contact with E. histolytica. The mean of 3 biological replicates for each experimental condition relative to the mean of untreated wild type cells is shown; error bars represent the s.e.m. (*P < 0.05; **P < 0.005; ***P < 0.001 calculated by two-tailed t test).
Mentions: We monitored extracellular levels of K+ levels and LDH in host cells exposed to E. histolytica over an hour. We found that increased extracellular K+ preceded elevated LDH, suggesting that K+ efflux occurred prior to cell death (Fig. 6a). To further determine if E. histolytica induced efflux of intracellular K+, cells were loaded with the K+-sensitive fluorescent intravital dye PBFI. Changes in intracellular K+ concentrations in HT-29 cells during contact with E. histolytica were monitored over 30 minutes in M199 media (KCl = 5.33 mM64). Cells co-incubated with E. histolytica displayed a significant reduction in intracellular K+ after 30 minutes (Fig. 6b). The extracellular ionic concentrations of K+ in the supernatants of HT-29 cells were measured in parallel and an increase of 0.1 mM K+ in the presence of E. histolytica was detected after 30 minutes. No increase in extracellular K+ concentration was detected in the absence of E. histolytica or in E. histolytica alone (data not shown).

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