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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

Kcn gene expression and regulation in the human colon in amebiasis.Average gene expression (day 60) and regulation during acute amebiasis (Δ day 1–day 60) of the 94 annotated Kcn genes. Kcn was omitted from K+ channel gene names for clarity. Slc24a3 and Cftr are shown for reference. Kcn genes with documented intestinal expression are in blue. Genes that were significantly differentially regulated in disease are shown in red. Significance was determined as P < 0.001 by 2-way ANOVA of matched day 1 and day 60 samples (n = 8). Quadrants indicated the mean of expression (day 60) and regulation (Δ day 1–day 60) of all probes on the microarray. Normal expression (day 60) was significantly correlated with the regulation during disease (w day 1–day 60) by linear regression (n = 8 matched samples, R = −0.54 P > 0.0001.
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f4: Kcn gene expression and regulation in the human colon in amebiasis.Average gene expression (day 60) and regulation during acute amebiasis (Δ day 1–day 60) of the 94 annotated Kcn genes. Kcn was omitted from K+ channel gene names for clarity. Slc24a3 and Cftr are shown for reference. Kcn genes with documented intestinal expression are in blue. Genes that were significantly differentially regulated in disease are shown in red. Significance was determined as P < 0.001 by 2-way ANOVA of matched day 1 and day 60 samples (n = 8). Quadrants indicated the mean of expression (day 60) and regulation (Δ day 1–day 60) of all probes on the microarray. Normal expression (day 60) was significantly correlated with the regulation during disease (w day 1–day 60) by linear regression (n = 8 matched samples, R = −0.54 P > 0.0001.

Mentions: The primary RNAi screen identified ion channels as mediators of cell killing in the bladder epithelial cell line UMUC3. Ion channels and their regulatory subunits exhibit tissue specific expression50. To establish the role of ion transport in colonic amebic infection we examined the expression of K+ channel (Kcn) genes during E. histolytica infection of the human colon37 (Fig. 4). We compared expression of the 94 annotated Kcn genes during acute amebic colitis in humans to matched biopsies from recovered patients. Kcn genes were globally down-regulated during acute amebiasis (day 1 of acute amebiasis). Furthermore, the level of down-regulation correlated with the degree of expression in the colon in recovered patients (60 days post infection expression) (R = −0.54, P < 0. 0001). The Cl− channel Cftr was also highly expressed in the colon and was down-regulated during acute amebiasis. Down-regulation of colonic Kcn genes and Cftr may be a protective physiological response to prevent excessive luminal secretion and cell death during amebiasis. Additionally, cells with higher Kcn gene expression may be more sensitive to E. histolytica killing leading to an overall depletion of Kcn transcripts in the colon during amebic infection.


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)

Kcn gene expression and regulation in the human colon in amebiasis.Average gene expression (day 60) and regulation during acute amebiasis (Δ day 1–day 60) of the 94 annotated Kcn genes. Kcn was omitted from K+ channel gene names for clarity. Slc24a3 and Cftr are shown for reference. Kcn genes with documented intestinal expression are in blue. Genes that were significantly differentially regulated in disease are shown in red. Significance was determined as P < 0.001 by 2-way ANOVA of matched day 1 and day 60 samples (n = 8). Quadrants indicated the mean of expression (day 60) and regulation (Δ day 1–day 60) of all probes on the microarray. Normal expression (day 60) was significantly correlated with the regulation during disease (w day 1–day 60) by linear regression (n = 8 matched samples, R = −0.54 P > 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Kcn gene expression and regulation in the human colon in amebiasis.Average gene expression (day 60) and regulation during acute amebiasis (Δ day 1–day 60) of the 94 annotated Kcn genes. Kcn was omitted from K+ channel gene names for clarity. Slc24a3 and Cftr are shown for reference. Kcn genes with documented intestinal expression are in blue. Genes that were significantly differentially regulated in disease are shown in red. Significance was determined as P < 0.001 by 2-way ANOVA of matched day 1 and day 60 samples (n = 8). Quadrants indicated the mean of expression (day 60) and regulation (Δ day 1–day 60) of all probes on the microarray. Normal expression (day 60) was significantly correlated with the regulation during disease (w day 1–day 60) by linear regression (n = 8 matched samples, R = −0.54 P > 0.0001.
Mentions: The primary RNAi screen identified ion channels as mediators of cell killing in the bladder epithelial cell line UMUC3. Ion channels and their regulatory subunits exhibit tissue specific expression50. To establish the role of ion transport in colonic amebic infection we examined the expression of K+ channel (Kcn) genes during E. histolytica infection of the human colon37 (Fig. 4). We compared expression of the 94 annotated Kcn genes during acute amebic colitis in humans to matched biopsies from recovered patients. Kcn genes were globally down-regulated during acute amebiasis (day 1 of acute amebiasis). Furthermore, the level of down-regulation correlated with the degree of expression in the colon in recovered patients (60 days post infection expression) (R = −0.54, P < 0. 0001). The Cl− channel Cftr was also highly expressed in the colon and was down-regulated during acute amebiasis. Down-regulation of colonic Kcn genes and Cftr may be a protective physiological response to prevent excessive luminal secretion and cell death during amebiasis. Additionally, cells with higher Kcn gene expression may be more sensitive to E. histolytica killing leading to an overall depletion of Kcn transcripts in the colon during amebic infection.

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