Phage-encoded Shiga Toxin as a Bacterial Defense Against Predation: Shiga Toxin Induction, Uptake and Toxicity in Tetrahymena Thermophila
Gerald Koudelka Principal Investigator
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Intellectual merit<br/>Exotoxins of bacteria are among the most deadly substances known. These exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. When studied in the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. The genes encoding these toxins are usually encoded by viruses of bacteria (bacteriophages). Environmental DNA sequencing (metagenomic analyses) indicates that ~15% of all bacteriophage (both free and incorporated into bacteria) encode an exotoxin gene. With approximately 106 bacteriophages /mL of seawater and 108 bacteriophages/gram of sediment, the frequency of occurrence of the genes encoding any particular exotoxin gene in bacteriophage far exceeds the number of potential animal hosts. Moreover, these bacteriophage-encoded exotoxin genes are found at high frequency in phages isolated from environments where the corresponding human diseases are not prevalent. These observations suggest the hypothesis that humans and other susceptible mammals are neither the original nor primary targets of these toxins. Hence any anthropogenic effects on the evolution of these toxins may not be as significant as those that occurred before the dawn of man and other multicellular organisms. <br/>If not mammals, then "who" are the true targets of the ubiquitous exotoxins. A major source of bacterial mortality is consumption by single-celled eukaryotic predators, such as ciliates and other protozoa. Bacteria have evolved many different methods to fend off these predators, including becoming oversized and fleeing at high speeds. Coordinated release of phage-encoded exotoxins, either at the pre- or post-ingestional states via lytic induction of lysogenic bacteriophage comprises another possible antipredator defense strategy. Protozoa are phagocytotic cells and they share many features with mammalian phagocytes, especially macrophages. The phage-encoded exotoxins like the well-studied Shiga toxin (Stx), kill eukaryotic cells by attacking features and pathways that are common to all eukaryotes both, uni- and multi-cellular. Since predation by eukaryotic predators (e.g., ciliates and other protozoa), is a major source of bacterial mortality, these observations suggest that exotoxins may have arisen as part of an antipredator, (antiprotozoan) defense strategy. Thus the evolution of these toxins may have occurred before the appearance of multicellular organisms. Hence humans may be innocent bystanders in the evolutionary battle between protozoans and their bacterial prey.<br/>Using a naturally occurring predator-prey interaction, the studies in this project explore aspects of how bacteriophage-encoded exotoxins like Shiga toxin are used as part of the bacterial anti-predator arsenal. These studies will provide the molecular details of the bacterial response to predation by determining 1) how T. thermophila signals bacteria that are lysogenic for toxin-encoding phages to produce Shiga toxin and 2) how this exotoxin enters and causes cytotoxicity in these ciliates. Some of these mechanisms are apparently unique to the ciliates and others may be conserved in higher eukaryotes. <br/><br/>Broader Impacts<br/><br/>The proposed research will provide a hypothesis-driven research experience to both graduate and undergraduate students at the University at Buffalo institution and through collaborations, undergraduate students at Mercyhurst College, a small, liberal arts college located in Erie, PA. In the Koudelka lab, students have learned how to ask important biological questions and used their observations to make important contributions to scientific understanding. Dr. Koudelka has supervised >22 undergraduates and high school students in the last 15 years, >50% of which were members of underrepresented minority groups or women. All of the PI's trainees have gone on to successful careers in research or medicine. The results of these studies will help <br/>define how predation and bacterial responses to it, influence the evolution and structure of microbial communities.