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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

Validation of candidate susceptibility genes in a secondary screen.55 candidate genes were selected for validation by esiRNA. Cytotoxicity was normalized to FLUC controls for each knockdown. K+ channels are bolded. The means of triplicate experimental values were averaged for three independent experiments. The error bars represent the range of means in triplicate independent experiments. *P < 0.01 calculated for candidate genes relative to FLUC by one-way ANOVA Fisher’s LSD test (each comparison to FLUC stands alone).
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f2: Validation of candidate susceptibility genes in a secondary screen.55 candidate genes were selected for validation by esiRNA. Cytotoxicity was normalized to FLUC controls for each knockdown. K+ channels are bolded. The means of triplicate experimental values were averaged for three independent experiments. The error bars represent the range of means in triplicate independent experiments. *P < 0.01 calculated for candidate genes relative to FLUC by one-way ANOVA Fisher’s LSD test (each comparison to FLUC stands alone).

Mentions: Due to the high probability of off target effects in pooled shRNA screens we tested 55 candidate susceptibility genes in a secondary screen. In this screen each candidate gene was silenced with endoribonuclease-digested siRNA (esiRNA) and amebic cytotoxicity was directly measured. 35 of 55 candidate susceptibility genes reduced amebic cytotoxicity relative to the FLUC control. 15 reached significance in triplicate assays (1-way ANOVA, P < 0.05). 9 knockdowns had marginal effects on amebic killing (<5%) and 12 knockdowns increased susceptibility to amebic cytotoxicity, 7 of which reached significance (1-way ANOVA, P < 0.05) (Fig. 2). Overall, 65% of hits from the primary screen reduced amebic cytotoxicity in the secondary screen, including Flrt3, the gene with the highest evidence score. Flrt3 knockdown reduced amebic killing by 20% relative to controls. It is of note that K+ ion channels that were not validated (Kcnip4 and Slc24a3) had evidence scores of <2, while the K+ channels with higher evidence scores (Kcnb2, Kcna3, Kcnj3) significantly reduced amebic killing by ~16% (P < 0.05) (Fig. 2 and Table 3).


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)

Validation of candidate susceptibility genes in a secondary screen.55 candidate genes were selected for validation by esiRNA. Cytotoxicity was normalized to FLUC controls for each knockdown. K+ channels are bolded. The means of triplicate experimental values were averaged for three independent experiments. The error bars represent the range of means in triplicate independent experiments. *P < 0.01 calculated for candidate genes relative to FLUC by one-way ANOVA Fisher’s LSD test (each comparison to FLUC stands alone).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Validation of candidate susceptibility genes in a secondary screen.55 candidate genes were selected for validation by esiRNA. Cytotoxicity was normalized to FLUC controls for each knockdown. K+ channels are bolded. The means of triplicate experimental values were averaged for three independent experiments. The error bars represent the range of means in triplicate independent experiments. *P < 0.01 calculated for candidate genes relative to FLUC by one-way ANOVA Fisher’s LSD test (each comparison to FLUC stands alone).
Mentions: Due to the high probability of off target effects in pooled shRNA screens we tested 55 candidate susceptibility genes in a secondary screen. In this screen each candidate gene was silenced with endoribonuclease-digested siRNA (esiRNA) and amebic cytotoxicity was directly measured. 35 of 55 candidate susceptibility genes reduced amebic cytotoxicity relative to the FLUC control. 15 reached significance in triplicate assays (1-way ANOVA, P < 0.05). 9 knockdowns had marginal effects on amebic killing (<5%) and 12 knockdowns increased susceptibility to amebic cytotoxicity, 7 of which reached significance (1-way ANOVA, P < 0.05) (Fig. 2). Overall, 65% of hits from the primary screen reduced amebic cytotoxicity in the secondary screen, including Flrt3, the gene with the highest evidence score. Flrt3 knockdown reduced amebic killing by 20% relative to controls. It is of note that K+ ion channels that were not validated (Kcnip4 and Slc24a3) had evidence scores of <2, while the K+ channels with higher evidence scores (Kcnb2, Kcna3, Kcnj3) significantly reduced amebic killing by ~16% (P < 0.05) (Fig. 2 and Table 3).

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