Phage Encoded Toxins as a Bacterial Defense against Single Celled Eukaryotic Predators
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Bacterially-derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found on bacterial viruses (bacteriophages). When studied in the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-encoded exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian targets, suggesting mammals are not the primary 'targets' of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that Tetrahymena thermophila, a bacterivorous predator, are killed when co-cultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In co-cultures with Tetrahymena, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx. Tetrahymena are also killed by purified Stx. Disruption of the gene encoding the StxB subunit or adding an excess of the non-toxic StxB subunit substantially reduced Stx holotoxin toxicity suggesting that this subunit mediates intake and/or trafficking of Stx by Tetrahymena. Bacterially-mediated Tetrahymena killing was blocked by mutations that prevented the bacterial SOS response ( recA -) or by enzymes that breakdown H 2 O 2 (catalase), suggesting that the production of H 2 O 2 by Tetrahymena signals its presence to presence to the bacteria, leading to bacteriophage induction and production of Stx. Stx toxicity in mammalian cells results from Stx binding to its glycosphingolipid receptor globotriaosyl ceramide (Gb3), and enters into the cytoplasm via retrograde transport and inactivates the ribosome by its glycosidase activity. We show here that shiga toxin toxicity to T. thermophila is decreased when competed with excess shiga toxin B subunit (StxB) or Ricin B, the carbohydrate binding portion of Ricin. Hence our data suggest that Stx enters T. thermophila by a glycoconjugate receptor. We also show that Stx can enter and kill Tetrahymena when it is produced by bacteria outside the Tetrahymena cell or when produced by bacteria encapsulated within food vacuoles. Protection against killing by internally and externally produced Stx can be effected by adding purified StxB, suggesting that these seemingly different pathways are actually the same. Incubation of Tetrahymena with purified shiga toxin decreases total protein synthesis suggesting this is its mechanism of killing Tetrahymena. Therefore, Stx apparently kills Tetrahymena in the same manner it does mammalian cells.