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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

Model for E. histolytica activation of K+ channels in host cell killing.In both intestinal epithelial and immune cells, E. histolytica triggers increased intracellular Ca2+ and cAMP. In an intestinal epithelial cell (purple cell on the right), Cl− efflux is mediated by apical Cftr, while K+ efflux occurs at both apical and basolateral surfaces. Increased intracellular Ca2+ activates large conductance K+ channels in the apical and basolateral membranes. Cl− efflux may be activated directly by increased intracellular Ca2+ and/or cAMP or may occur secondarily to K+ efflux to regulate cellular charge polarization. As K+ and Cl− efflux occurs, water and intracellular ion concentrations fall which causes cells to shrink. Cell shrinkage and decreased intracellular K+ trigger caspase activation. In intestinal epithelial cells it appears that caspase-3 is activated leading to apoptotic death. In macrophages (blue cell on the left), decreased cytosolic K+ concentration mediated caspase-1 activation leading to inflammasome activation, IL-1β secretion and pyroptotic cell death.
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f8: Model for E. histolytica activation of K+ channels in host cell killing.In both intestinal epithelial and immune cells, E. histolytica triggers increased intracellular Ca2+ and cAMP. In an intestinal epithelial cell (purple cell on the right), Cl− efflux is mediated by apical Cftr, while K+ efflux occurs at both apical and basolateral surfaces. Increased intracellular Ca2+ activates large conductance K+ channels in the apical and basolateral membranes. Cl− efflux may be activated directly by increased intracellular Ca2+ and/or cAMP or may occur secondarily to K+ efflux to regulate cellular charge polarization. As K+ and Cl− efflux occurs, water and intracellular ion concentrations fall which causes cells to shrink. Cell shrinkage and decreased intracellular K+ trigger caspase activation. In intestinal epithelial cells it appears that caspase-3 is activated leading to apoptotic death. In macrophages (blue cell on the left), decreased cytosolic K+ concentration mediated caspase-1 activation leading to inflammasome activation, IL-1β secretion and pyroptotic cell death.

Mentions: At the intestinal epithelium Cl− efflux is mediated by the apical Cftr (identified in our RNAi screen), while K+ efflux occurs at the apical and basolateral surface100101102. Intestinal cells are extremely sensitive to intracellular Ca2+ and cAMP concentration which activate K+ channels in luminal and basolateral membranes5152626970. A model of ion transport induced by E. histolytica via increased intracellular Ca2+ and cAMP levels is shown in Fig. 8. It remains to be determined if K+ and Cl− efflux are each triggered directly or if one occurs secondarily to balance charge across the membrane. In the event of K+ and Cl− efflux, intracellular ion concentrations fall, triggering water secretion and cell shrinkage leading to apoptotic volume decrease death (AVD). Cytosolic K+ concentration regulates caspase activation7778 and increased extracellular K+ and inhibitors of Ca2+-activated K+ channels blocked intrinsic and extrinsic apoptotic pathways79. Low intracellular K+ concentrations activated caspase-1 and pyroptotic cell death656667. We found that caspase-1 activation was blocked by high extracellular K+ and by specific inhibitors of voltage-gated K+ channels in macrophages (Fig. 7). Caspase inhibitors also reduced cell death in IECS in the apparent absence of the inflammasome. These data led us to hypothesize that decreased cytosolic K+ concentration is the common biochemical signal that mediates rapid activation of different caspases in multiple cell types by E. histolytica.


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)

Model for E. histolytica activation of K+ channels in host cell killing.In both intestinal epithelial and immune cells, E. histolytica triggers increased intracellular Ca2+ and cAMP. In an intestinal epithelial cell (purple cell on the right), Cl− efflux is mediated by apical Cftr, while K+ efflux occurs at both apical and basolateral surfaces. Increased intracellular Ca2+ activates large conductance K+ channels in the apical and basolateral membranes. Cl− efflux may be activated directly by increased intracellular Ca2+ and/or cAMP or may occur secondarily to K+ efflux to regulate cellular charge polarization. As K+ and Cl− efflux occurs, water and intracellular ion concentrations fall which causes cells to shrink. Cell shrinkage and decreased intracellular K+ trigger caspase activation. In intestinal epithelial cells it appears that caspase-3 is activated leading to apoptotic death. In macrophages (blue cell on the left), decreased cytosolic K+ concentration mediated caspase-1 activation leading to inflammasome activation, IL-1β secretion and pyroptotic cell death.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Model for E. histolytica activation of K+ channels in host cell killing.In both intestinal epithelial and immune cells, E. histolytica triggers increased intracellular Ca2+ and cAMP. In an intestinal epithelial cell (purple cell on the right), Cl− efflux is mediated by apical Cftr, while K+ efflux occurs at both apical and basolateral surfaces. Increased intracellular Ca2+ activates large conductance K+ channels in the apical and basolateral membranes. Cl− efflux may be activated directly by increased intracellular Ca2+ and/or cAMP or may occur secondarily to K+ efflux to regulate cellular charge polarization. As K+ and Cl− efflux occurs, water and intracellular ion concentrations fall which causes cells to shrink. Cell shrinkage and decreased intracellular K+ trigger caspase activation. In intestinal epithelial cells it appears that caspase-3 is activated leading to apoptotic death. In macrophages (blue cell on the left), decreased cytosolic K+ concentration mediated caspase-1 activation leading to inflammasome activation, IL-1β secretion and pyroptotic cell death.
Mentions: At the intestinal epithelium Cl− efflux is mediated by the apical Cftr (identified in our RNAi screen), while K+ efflux occurs at the apical and basolateral surface100101102. Intestinal cells are extremely sensitive to intracellular Ca2+ and cAMP concentration which activate K+ channels in luminal and basolateral membranes5152626970. A model of ion transport induced by E. histolytica via increased intracellular Ca2+ and cAMP levels is shown in Fig. 8. It remains to be determined if K+ and Cl− efflux are each triggered directly or if one occurs secondarily to balance charge across the membrane. In the event of K+ and Cl− efflux, intracellular ion concentrations fall, triggering water secretion and cell shrinkage leading to apoptotic volume decrease death (AVD). Cytosolic K+ concentration regulates caspase activation7778 and increased extracellular K+ and inhibitors of Ca2+-activated K+ channels blocked intrinsic and extrinsic apoptotic pathways79. Low intracellular K+ concentrations activated caspase-1 and pyroptotic cell death656667. We found that caspase-1 activation was blocked by high extracellular K+ and by specific inhibitors of voltage-gated K+ channels in macrophages (Fig. 7). Caspase inhibitors also reduced cell death in IECS in the apparent absence of the inflammasome. These data led us to hypothesize that decreased cytosolic K+ concentration is the common biochemical signal that mediates rapid activation of different caspases in multiple cell types by E. histolytica.

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