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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

Inhibition of K+ channel activity blocked amebic cytotoxicity in HT-29 IECs and amebic cytotoxicity and IL-1β production in THP-1 macrophages.(a) IL-1β secretion was measured by ELISA in HT-29 cells and differentiated THP-1 macrophages exposed to E. histolytica for 180 minutes (1 trophozoite to 5 cells). HT-29 cells did not secrete a detectable level of IL-1β (data not shown). Inhibitors did not cause LDH release in the absence of E. histolytica. Experimental values were normalized and expressed as the percent of vehicle treated controls, the mean and s.e.m. of three biological replicates is shown. P values were calculated relative to untreated cells (*P < 0.05; **P ≤ 0.008) by two-tailed Fisher’s LSD test. (b) LDH, IL-1β and cleaved caspase-1 in cells treated with KCl (50 mM), ChoCl (50 mM) AM92016 (10 μK) and YVAD (10 μ). Each experimental condition was normalized and is expressed relative to untreated wild type cells; the mean and s.e.m. of three biological replicates is shown. P values were calculated relative to untreated cells (*P ≤ 0.02; **P ≤ 0.005) by Fisher’s LSD test.
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f7: Inhibition of K+ channel activity blocked amebic cytotoxicity in HT-29 IECs and amebic cytotoxicity and IL-1β production in THP-1 macrophages.(a) IL-1β secretion was measured by ELISA in HT-29 cells and differentiated THP-1 macrophages exposed to E. histolytica for 180 minutes (1 trophozoite to 5 cells). HT-29 cells did not secrete a detectable level of IL-1β (data not shown). Inhibitors did not cause LDH release in the absence of E. histolytica. Experimental values were normalized and expressed as the percent of vehicle treated controls, the mean and s.e.m. of three biological replicates is shown. P values were calculated relative to untreated cells (*P < 0.05; **P ≤ 0.008) by two-tailed Fisher’s LSD test. (b) LDH, IL-1β and cleaved caspase-1 in cells treated with KCl (50 mM), ChoCl (50 mM) AM92016 (10 μK) and YVAD (10 μ). Each experimental condition was normalized and is expressed relative to untreated wild type cells; the mean and s.e.m. of three biological replicates is shown. P values were calculated relative to untreated cells (*P ≤ 0.02; **P ≤ 0.005) by Fisher’s LSD test.

Mentions: The NLRP3 inflammasome is activated by multiple stimuli and K+ efflux is reported to be the common trigger for diverse stimuli66. We found that E. histolytica inflammasome activation in THP-1 macrophages required K+ efflux as increased extracellular K+ and specific K+ channel inhibitors (AM 92016 and CP 339818) blocked IL-1β secretion and amebic cytotoxicity in THP-1 cells (Fig. 7a). We tested the effect of caspase inhibitors on inflammasome activation and killing of THP-1 cells by E. histolytica. Caspase-1 inhibition blocked both cell killing and IL-1β secretion. Caspase-3 inhibition blocked cell killing but not IL-1β secretion, while caspase-4 inhibition had no effect. The pan-caspase inhibitor zVAD-FMK significantly inhibited IL-1β secretion and amebic cytotoxicity in THP-1 cells (Fig. S2). Thus both caspase-1 and K+ efflux were required for IL-1β secretion in response to E. histolytica by THP-1 macrophages.


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)

Inhibition of K+ channel activity blocked amebic cytotoxicity in HT-29 IECs and amebic cytotoxicity and IL-1β production in THP-1 macrophages.(a) IL-1β secretion was measured by ELISA in HT-29 cells and differentiated THP-1 macrophages exposed to E. histolytica for 180 minutes (1 trophozoite to 5 cells). HT-29 cells did not secrete a detectable level of IL-1β (data not shown). Inhibitors did not cause LDH release in the absence of E. histolytica. Experimental values were normalized and expressed as the percent of vehicle treated controls, the mean and s.e.m. of three biological replicates is shown. P values were calculated relative to untreated cells (*P < 0.05; **P ≤ 0.008) by two-tailed Fisher’s LSD test. (b) LDH, IL-1β and cleaved caspase-1 in cells treated with KCl (50 mM), ChoCl (50 mM) AM92016 (10 μK) and YVAD (10 μ). Each experimental condition was normalized and is expressed relative to untreated wild type cells; the mean and s.e.m. of three biological replicates is shown. P values were calculated relative to untreated cells (*P ≤ 0.02; **P ≤ 0.005) by Fisher’s LSD test.
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Related In: Results  -  Collection

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f7: Inhibition of K+ channel activity blocked amebic cytotoxicity in HT-29 IECs and amebic cytotoxicity and IL-1β production in THP-1 macrophages.(a) IL-1β secretion was measured by ELISA in HT-29 cells and differentiated THP-1 macrophages exposed to E. histolytica for 180 minutes (1 trophozoite to 5 cells). HT-29 cells did not secrete a detectable level of IL-1β (data not shown). Inhibitors did not cause LDH release in the absence of E. histolytica. Experimental values were normalized and expressed as the percent of vehicle treated controls, the mean and s.e.m. of three biological replicates is shown. P values were calculated relative to untreated cells (*P < 0.05; **P ≤ 0.008) by two-tailed Fisher’s LSD test. (b) LDH, IL-1β and cleaved caspase-1 in cells treated with KCl (50 mM), ChoCl (50 mM) AM92016 (10 μK) and YVAD (10 μ). Each experimental condition was normalized and is expressed relative to untreated wild type cells; the mean and s.e.m. of three biological replicates is shown. P values were calculated relative to untreated cells (*P ≤ 0.02; **P ≤ 0.005) by Fisher’s LSD test.
Mentions: The NLRP3 inflammasome is activated by multiple stimuli and K+ efflux is reported to be the common trigger for diverse stimuli66. We found that E. histolytica inflammasome activation in THP-1 macrophages required K+ efflux as increased extracellular K+ and specific K+ channel inhibitors (AM 92016 and CP 339818) blocked IL-1β secretion and amebic cytotoxicity in THP-1 cells (Fig. 7a). We tested the effect of caspase inhibitors on inflammasome activation and killing of THP-1 cells by E. histolytica. Caspase-1 inhibition blocked both cell killing and IL-1β secretion. Caspase-3 inhibition blocked cell killing but not IL-1β secretion, while caspase-4 inhibition had no effect. The pan-caspase inhibitor zVAD-FMK significantly inhibited IL-1β secretion and amebic cytotoxicity in THP-1 cells (Fig. S2). Thus both caspase-1 and K+ efflux were required for IL-1β secretion in response to E. histolytica by THP-1 macrophages.

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