Limits...
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

Design and implementation of a whole genome RNAi screen to identify host factors required for amebic cytotoxicity(a) Screening of the RNAi library with E. histolytica parasites.After each selection, the parasites were removed and resistant cells were expanded and selected successively at a ratio of 1 parasite to 5 host cells, except for the ninth and final round of selection, (1 parasite to 2 host cells). (b) Selection increased resistance to amebic killing. Pool 6 of the RNAi library pool exhibited increased resistance relative to an empty vector pool selected in parallel after 6 rounds of selection. In the ninth and final round of selection each library was selected until no surviving cells were visible by microscopic examination. Percent survival of UMUC3 cells was determined by visual assessment of the monolayers after selection. Cells that survived round 9 of selection (pool 9) were sequenced to identify candidate genes. (c) The selected RNAi library (RNAi) and the empty vector (EV) control from pool 9 of the screen were assayed for resistance to amebic killing. The mean of biological triplicates and s.e.m. is shown and analyzed by 2-tailed t test (*P < 0.01, **P < 0.001) (d) Bioinformatics analysis of candidate susceptibility genes. The top overrepresented biological processes are shown with their enrichment value. (e) Clusters of ion transport associated processes in candidate genes. Clusters were based on similarity (kappa statistic >0.3). The percent of genes in each cluster is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4561901&req=5

f1: Design and implementation of a whole genome RNAi screen to identify host factors required for amebic cytotoxicity(a) Screening of the RNAi library with E. histolytica parasites.After each selection, the parasites were removed and resistant cells were expanded and selected successively at a ratio of 1 parasite to 5 host cells, except for the ninth and final round of selection, (1 parasite to 2 host cells). (b) Selection increased resistance to amebic killing. Pool 6 of the RNAi library pool exhibited increased resistance relative to an empty vector pool selected in parallel after 6 rounds of selection. In the ninth and final round of selection each library was selected until no surviving cells were visible by microscopic examination. Percent survival of UMUC3 cells was determined by visual assessment of the monolayers after selection. Cells that survived round 9 of selection (pool 9) were sequenced to identify candidate genes. (c) The selected RNAi library (RNAi) and the empty vector (EV) control from pool 9 of the screen were assayed for resistance to amebic killing. The mean of biological triplicates and s.e.m. is shown and analyzed by 2-tailed t test (*P < 0.01, **P < 0.001) (d) Bioinformatics analysis of candidate susceptibility genes. The top overrepresented biological processes are shown with their enrichment value. (e) Clusters of ion transport associated processes in candidate genes. Clusters were based on similarity (kappa statistic >0.3). The percent of genes in each cluster is shown.

Mentions: The input pooled shRNA library was subjected to successive rounds of selection with E. histolytica trophozoites. After each round of selection, resistant cells were separated from trophozoites and cultured to obtain a sufficient cell number for rescreening. Samples were taken after every round of selection to track the loss of susceptible clones (Fig. 1a). The RNAi library had increased resistance to E. histolytica cytotoxicity relative to the empty vector control library screened in parallel after 6 rounds of selection (Fig. 1b). The screen was continued for three additional rounds of selection, with the final round of selection at the higher ratio of 1:2 parasites to host cells.


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)

Design and implementation of a whole genome RNAi screen to identify host factors required for amebic cytotoxicity(a) Screening of the RNAi library with E. histolytica parasites.After each selection, the parasites were removed and resistant cells were expanded and selected successively at a ratio of 1 parasite to 5 host cells, except for the ninth and final round of selection, (1 parasite to 2 host cells). (b) Selection increased resistance to amebic killing. Pool 6 of the RNAi library pool exhibited increased resistance relative to an empty vector pool selected in parallel after 6 rounds of selection. In the ninth and final round of selection each library was selected until no surviving cells were visible by microscopic examination. Percent survival of UMUC3 cells was determined by visual assessment of the monolayers after selection. Cells that survived round 9 of selection (pool 9) were sequenced to identify candidate genes. (c) The selected RNAi library (RNAi) and the empty vector (EV) control from pool 9 of the screen were assayed for resistance to amebic killing. The mean of biological triplicates and s.e.m. is shown and analyzed by 2-tailed t test (*P < 0.01, **P < 0.001) (d) Bioinformatics analysis of candidate susceptibility genes. The top overrepresented biological processes are shown with their enrichment value. (e) Clusters of ion transport associated processes in candidate genes. Clusters were based on similarity (kappa statistic >0.3). The percent of genes in each cluster is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Design and implementation of a whole genome RNAi screen to identify host factors required for amebic cytotoxicity(a) Screening of the RNAi library with E. histolytica parasites.After each selection, the parasites were removed and resistant cells were expanded and selected successively at a ratio of 1 parasite to 5 host cells, except for the ninth and final round of selection, (1 parasite to 2 host cells). (b) Selection increased resistance to amebic killing. Pool 6 of the RNAi library pool exhibited increased resistance relative to an empty vector pool selected in parallel after 6 rounds of selection. In the ninth and final round of selection each library was selected until no surviving cells were visible by microscopic examination. Percent survival of UMUC3 cells was determined by visual assessment of the monolayers after selection. Cells that survived round 9 of selection (pool 9) were sequenced to identify candidate genes. (c) The selected RNAi library (RNAi) and the empty vector (EV) control from pool 9 of the screen were assayed for resistance to amebic killing. The mean of biological triplicates and s.e.m. is shown and analyzed by 2-tailed t test (*P < 0.01, **P < 0.001) (d) Bioinformatics analysis of candidate susceptibility genes. The top overrepresented biological processes are shown with their enrichment value. (e) Clusters of ion transport associated processes in candidate genes. Clusters were based on similarity (kappa statistic >0.3). The percent of genes in each cluster is shown.
Mentions: The input pooled shRNA library was subjected to successive rounds of selection with E. histolytica trophozoites. After each round of selection, resistant cells were separated from trophozoites and cultured to obtain a sufficient cell number for rescreening. Samples were taken after every round of selection to track the loss of susceptible clones (Fig. 1a). The RNAi library had increased resistance to E. histolytica cytotoxicity relative to the empty vector control library screened in parallel after 6 rounds of selection (Fig. 1b). The screen was continued for three additional rounds of selection, with the final round of selection at the higher ratio of 1:2 parasites to host cells.

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