Team:ETH Zurich/Circuit/Fd Heat Sensor
Heat Sensor
Introduction
Sensing the temperature is crucial for the lifecycle of many bacteria and virus. We adapt this function from a specific heat-inducible system and engineer it to fit our needs for activating the toxin release in CATE.
Various thermosensitive operator systems exist in nature and they differ massively in their way of function. Four general classes of thermosensors exist: DNA, RNA, protein or lipid-protein thermosensors. DNA thermosensors rely on the bending of DNA at lower temperatures, which enables cooperative binding of DNA-associated proteins. RNA based thermosensors form a stem-loop in the messenger RNA, which hides the Shine-Dalgarno sequence and the AUG translation initiation codon. At higher temperatures, the hydrogen bonds of the stem-loop break apart and the ribosomal subunits can associate with the RNA and initiate translation. While DNA-and RNA thermosensors act before translation, for protein mediated temperature sensing, a translated protein needs to be present in the cytoplasm. This comes with the potential to tune the translation initiation rate by changing the ribosome binding site affinity to the ribosome.
The delivery method
For CATE, we chose the TlpA thermosensor, which is derived from Salmonella and belongs to the protein thermosensors. The advantage of this system is the high on/off-ratio of up to 300 and induction temperature of 45 °C, a temperature not reached by fever, but still below levels that cause damage in tissues surrounding a tumor. We adapt this function so that we can use it to trigger the release of the Anti-Cancer Toxin.
The TlpA system consists of a constitutively expressed regulator protein called TlpA and an inducible TlpA operator-promotor called PTlpA. TlpA contains an approximately 300-residue coiled-coil domain at the C-terminus that uncoils between 42 °C and 45 °C. In low temperatures, its N-terminal domain is in a dimeric state and can bind the 52-bp PTlpA. Transcription of the downstream gene can therefore happen at temperatures above 42 °C but not below. [Piraner et al.]
The delivery method
The release of the Anti-Cancer Toxin from our system is designed to happen once both Checkpoint 1 and Checkpoint 2 have been passed. This means that the toxin has successfully been accumulated inside of the bacteria and is ready to be delivered to the tumor. Considering that simultaneous release of the full dose of the toxin is desirable, we decided that synchronized bacterial cell lysis should be our method of choice.
The lytic agent
Protein E, our weapon of choice, is a protein produced by phage Phi X 147, which (in nature) lyses the host cell after production of phage particles. [2] We decided to use it because it has already been successfully implemented to achieve cell lysis in engineered bacteria [3] and it was kindly provided to us by Dr. Irene Wuthrich from our department, along with advice on how to work with it.
The exact mechanism of action of protein E has long been controversial and different models were proposed to explain its lytic function. It has been suggested that protein E activates a component of the E. coli autolytic system, that it inhibits cell wall synthesis in a manner similar to penicillin or that it oligomerizes to form a transmembrane tunnel, all leading to release of cytoplasmic content and ultimately cell death. [4][5][6]
However, it is now generally accepted that the most probable cellular target of protein E is an enzyme called Translocase I, encoded by the mraY gene. Translocase I seems to play an important role for cell wall biosynthesis and its inhibition leads to cell lysis. The "transmembrane tunnel" model has largely been discarded and the tunnels are now attributed to faulty cell wall synthesis, making it a consequence rather than the cause. [4]
Protein E and CATE
In our design, protein E is the mediator of cell lysis. Once the Anti-Cancer Toxin has accumulated and the MRI Contrast Agent has signalled passage through Checkpoint 1, the physician can apply focused ultrasound to activate the Heat Sensor. Activation of the Heat Sensor leads to production of protein E, which quickly results in cell lysis and toxin release.
References
- ^ Forbes, Neil S. "Engineering the perfect (bacterial) cancer therapy." Nature reviews. Cancer 10.11 (2010): 785.
- ^ Bernhardt, Thomas G., William D. Roof, and Ry Young. "Genetic evidence that the bacteriophage φX174 lysis protein inhibits cell wall synthesis." Proceedings of the National Academy of Sciences 97.8 (2000): 4297-4302.
- ^ Din, M. Omar, et al. "Synchronized cycles of bacterial lysis for in vivo delivery." Nature 536.7614 (2016): 81-85.
- ^ Roof, William D., and R. Young. "Phi X174 E complements lambda S and R dysfunction for host cell lysis." Journal of bacteriology 175.12 (1993): 3909-3912.
- ^ Lubitz, W., R. E. Harkness, and E. E. Ishiguro. "Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174." Journal of bacteriology 159.1 (1984): 385-387.
- ^ Witte, Angela, et al. "Endogenous transmembrane tunnel formation mediated by phi X174 lysis protein E." Journal of bacteriology 172.7 (1990): 4109-4114.
