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 in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian “targets,” suggesting that 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, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with Tetrahymena , the Stx-encoding bacteria display a growth advantage over those that do not produce Stx. Tetrahymena is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic 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 mutations) 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 the bacteria, leading to bacteriophage induction and production of Stx.

In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and aquatic ecosystems. We then relate the abundance of Shiga toxin in the environment to work that demonstrates that the genetic mechanisms underlying the production of Shiga toxin genes are modified and embellished beyond the classical microbial gene regulatory paradigms in a manner that apparently “fine tunes” the trigger to modulate the amount of toxin produced. Last, we highlight several recent studies examining microbe/protist interactions that postulate an answer to the outstanding question of why microbes might harbor and express Shiga toxin genes in the environment.

The affinities of the bacteriophage 434 repressor for its various binding sites depend on the type and/or concentration of monovalent cations. The ability of bacteriophage 434 repressor to govern the lysis-lysogeny decision depends on the DNA binding activities of the phage's c I repressor protein. We wished to determine whether changes in the intracellular ionic environment influence the lysis-lysogeny decision of the bacteriophage λ ^imm434 . Our findings show that the ionic composition within bacterial cells varies with the cation concentration in the growth media. When λ ^imm434 lysogens were grown to mid-log or stationary phase and subsequently incubated in media with increasing monovalent salt concentrations, we observed a salt concentration-dependent increase in the frequency of bacteriophage spontaneous induction. We also found that the frequency of spontaneous induction varied with the type of monovalent cation in the medium. The salt-dependent increase in phage production was unaffected by a recA mutation. These findings indicate that the salt-dependent increase in phage production is not caused by activation of the SOS pathway. Instead, our evidence suggests that salt stress induces this lysogenic bacteriophage by interfering with 434 repressor-DNA interactions. We speculate that the salt-dependent increase in spontaneous induction is due to a direct effect on the repressor's affinity for DNA. Regardless of the precise mechanism, our findings demonstrate that salt stress can regulate the phage lysis-lysogeny switch.

Phage-encoded Shiga toxin (Stx) acts as a bacterial defense against the eukaryotic predator Tetrahymena thermophila . It is unknown how Stx enters Tetrahymena protozoa or how it kills them. Tetrahymena protozoa are phagocytotic; hence, Stx could gain entry to the cytoplasm through the oral apparatus or via endocytosis. We find that Stx2 can kill T. thermophila protozoa that lack an oral apparatus, indicating that Stx2 can enter these cells via endocytosis. As opposed to the lack of effect on mammalian phagocytes, Stx2 produced by bacteria encapsulated within phagocytotic vesicles is also capable of killing Tetrahymena . Addition of an excess of the carbohydrate binding subunits of Stx2 (StxB) and/or ricin (ricin B) blocks Stx2 cytotoxicity. Thus, regardless of whether Stx2 enters the cytoplasm by endocytosis or from the phagocytotic vesicle, this transport is mediated by a putative glycoconjugate receptor. Bacteriophage-mediated lysis of Stx-encoding bacteria is necessary for Stx toxicity in Tetrahymena ; i.e., toxin released as a consequence of digestion of bacteria by Tetrahymena is harmless to the cell. This finding provides a rationale as to why the genes encoding Stx are found almost exclusively on bacteriophages; Stx must be released from the bacteria prior to the digestion of the cell, or it will not be able to exert its cytotoxic effect. It also suggests a reason why other bacterial exotoxins are also found only on temperate bacteriophages. Incubation of Tetrahymena with purified Stx2 decreases total protein synthesis. This finding indicates that, similar to mammalian cells, Stx2 kills Tetrahymena by inactivating its ribosomes. IMPORTANCE Tetrahymena is a bacterial predator and a model for mammalian phagocytosis and intracellular vesicular trafficking. Phage-encoded exotoxins apparently have evolved for the purpose of bacterial antipredator defense. These exotoxins kill mammalian cells by inactivating universally conserved factors and/or pathways. Tetrahymena and susceptible mammalian cells are killed when exposed to bacteriophage-encoded Shiga toxin (Stx). Stx toxicity in mammalian cells requires Stx binding to the globotriaosyl ceramide (Gb3) receptor, followed by receptor-mediated endocytosis (RME). We show that, similar to mammalian cells, internalized Stx inhibits protein synthesis in Tetrahymena . Although Tetrahymena lacks Gb3, our results suggest that the cytotoxic effect of Stx on Tetrahymena is apparently mediated by a receptor, thereby arguing for the existence of RME in Tetrahymena . As opposed to the case with mammalian phagocytes, Stx produced by bacteria inside Tetrahymena is cytotoxic, suggesting that these cells may represent a “missing link” between unicellular eukaryotic bacterial predators and phagocytotic mammalian cells.

Shiga toxins (Stx) induce the symptoms of the life-threatening hemolytic uremic syndrome (HUS) and are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC). The bacterial SOS response is the essential signal for high level production and release of Stx1/2. To assess the potential effectiveness of different antibiotics in blocking SOS response and Stx1/2 production, we constructed a reporter gene based test system that allows for the time-resolved, simultaneous read-out of the SOS response (recAP-cfp) and Stx1 production (stx1::yfp) in EHEC O157:H7 EDL933. We find that cells exposed to inhibitory or subinhibitory concentrations of ciprofloxacin did induce the SOS response, but not when the cells were exposed to rifaximine, azithromycin, tetracycline, gentamicin or ampicillin. Cell lysis and the peak in Stx1 production were substantially delayed with respect to the peak of the SOS response. We used this feature to show that adding transcriptional or translational inhibitors can block Stx1 production even after the SOS response is fully induced. RT-qPCR based tests with other clinically relevant EHEC isolates showed similar results for both Stx1 and Stx2. These observations suggest that transcriptional and translational inhibitors may be of value in treating EHEC infections.