Pseudomonas exotoxin kills Drosophila S2 cells via apoptosis.
Pseudomonas exotoxin A (PE) is cytotoxic for eukaryotic cells because it enters cells by receptor-mediated endocytosis, translocates to the cell cytosol and ADP-ribosylates elongation factor 2 (EF2). However, the interaction of this toxin with eukaryotic cells and the mechanism of PE-mediated cell death have not been extensively characterized. The feasibility of carrying out a genome-wide RNAi screen, makes Drosophila melanogaster S2 cells as a good model system to identify essential genes in PE-mediated cytotoxicity, provided a suitable multi-well assay is developed. Here, using the alamarBlue viability assay, we show that Drosophila S2 cells are sensitive to PE at picomolar concentrations and that toxin treatments provoke an increase in caspase activity. This prompted us to use RNAi to characterize the mechanism of cell death. Results indicated that PE-mediated death of S2 cells was dependent on the presence of diphthamide, the post translational modification of EF2, and on the presence of Drice, the terminal caspase of insect cells. RNAi to drice or chemical inhibition of caspase action by z-VAD-fmk protected cells from PE-mediated death. Protection from death by RNAi or z-VAD-fmk did not interfere with toxin delivery to the cytosol leading to inhibition of protein synthesis. Using a convenient alamarBlue assay, our data confirms the cytotoxicity of PE for S2 cells and establishes apoptosis as the mode of PE-mediated death. This confirms the suitability of Drosophila cells as a convenient and simple model to elucidate the role of specific genes and proteins required for PE action.
Sharma
Ashima K
AK
Biotherapy Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, HHS, 37 Convent Dr, Bethesda, MD 20892, USA.
FitzGerald
David
D
D036002:ADP Ribose Transferases / Q000494:pharmacology*; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001427:Bacterial Toxins / Q000494:pharmacology*; D001681:Biological Assay; D020169:Caspases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004330:Drosophila / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D029721:Drosophila Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005098:Exotoxins / Q000494:pharmacology*; D020868:Gene Silencing; D006639:Histidine / Q000031:analogs & derivatives / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D037521:Virulence Factors / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000590:Amino Acid Chloromethyl Ketones; D001427:Bacterial Toxins; D020169:Caspases; D029721:Drosophila Proteins; D005098:Exotoxins; D006639:Histidine; D034741:RNA, Small Interfering; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C027527:diphthamide; C109171:drICE protein, Drosophila