Different heat sensors

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. At lower temperatures, the DNA binding proteins can cover the operator region and prevent transcription. At higher temperatures, the DNA unbends and the operator region can promote transcription of the downstream genes.

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 and is a reason why we use a protein based thermosensor.

CATE's heat sensor

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 P_TlpA. 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 P_TlpA. Transcription of the downstream gene can therefore happen at temperatures above 42 °C but not below. [1][2]

The heat sensor is the safety Checkpoint 2

In a cancer treatment with CATE, the bacteria will prepare the Anti-Cancer Toxin in the cytoplasm together with the MRI Contrast Agent as soon as they sense a tumor environment with the Tumor Sensor. But the toxic compound is not released yet, because CATE might have colonized a wrong location, which also had the same environmental cues as a tumor (which is very unlikely). At this point the patient is analyzed in an MRI and a doctor can decide if the cancer toxin should be released. This is an additional safety checkpoint that is unprecedented for systemically (to the whole body) applied cancer therapeutics.

If the doctor decides to proceed with the treatment, he can use Focused Ultrasound, a technology already included in modern MRI devices, which can heat up patients tissue in a precisely defined area inside the body. Normally used with very high temperatures to destroy tumors, it also damages the surrounding healthy tissue, because the heat diffuses out of tumors. To prevent this off-target damage, we use the same Focused Ultrasound technology at a lower temperature, right high enough for CATE to be able to sense a change. The doctor will focus the ultrasound waves with the device to the area where the tumor is and heat the tumor tissue to 45 °C for up to 3 h. The heat sensor notices this change in temperature by unfolding the coiled-coil domains of the TlpA dimers. As a consequence, the dimers fall apart to monomers, which will not bind the P_TlpA anymore. The P_TlpA in an unbound state has a strong promotor activity and induces expression of protein E, which leads to Cell Lysis.

The Heat Sensor is activated by the physician via focused ultrasound which increases the temperature locally to 45 ^oC. This releases repression of P_TlpA by the repressor protein TlpA and, consequentially, expression of protein E is initiated. Protein E causes cell lysis by interfering with cell wall synthesis. Following cell lysis, azurin is released to the environment.