Mechanisms of Tetrodotoxin Synthesis in Microbial Symbionts - PROJECT SUMMARY Tetrodotoxin (TTX) is an extremely potent neurotoxin that gives pufferfish (fugu) sushi an element of danger. TTX derives its lethal toxicity from its ability to block the pore of almost all voltage-gated sodium channels, preventing most neurons and muscle cells from generating action potentials. Since its initial discovery in pufferfish, TTX has been identified in a wide array of animals, all of which appear to derive their TTX from bacteria, either directly or through sequestration from diet. Although the structure and mechanism of action of TTX were identified more than 50 years ago, the means by which it is synthesized in vivo are completely unknown. The long-term goal of this project is to elucidate the mechanisms of TTX biosynthesis, enabling development of assays for TTX, improving food safety, and facilitating the discovery of TTX analogs with therapeutic potential. To do so, we are leveraging our discovery of TTX-producing (TTX+) bacteria on the skin of newts that are endemic to the west coast of North America. These animals live in readily accessible freshwater habitats and are not threatened or endangered, allowing us to collect newts easily and isolate new strains of TTX+ bacteria. This ability is important, as previous researchers have been restricted to working with single strains of TTX+ bacteria isolated from marine animals, limiting attempts to uncover the genetic basis of TTX production. We plan to use three complementary approaches to discover genes involved in TTX biosynthesis. In Specific Aim 1, we will sequence complete genomes of all TTX+ bacteria that we have identified to date, comprising 22 strains across 7 genera. We will then analyze these genomes to identify candidate genes that may be involved in TTX synthesis. In Specific Aim 2, we will use experimental evolution to identify genes that are mutated, inverted, excised, or have decreased expression in strains that lose TTX production upon repeated passaging, a proclivity that has been previously described by other groups. In Specific Aim 3, we will use a forward genetic screen to create and identify mutants that do not produce TTX (TTX-). As an important component of this work, we will develop a high-throughput assay for detecting TTX; we are currently exploring two approaches, one involving MALDI-MS and the other involving a TTX-specific aptamer. We will determine which assay is more efficient and sensitive but will employ our more standard HILIC-MS/MS approach if both efforts fail. The genomes generated in Aim 1 will be used as a reference to identify the genes in which mutations reduce TTX production in our genetic screens in Aims 2 and 3. In tandem with this work we will develop protocols for genetic manipulation of our strains of interest to facilitate future work in which we will create knockouts to validate the candidate genes identified in all three aims.
