Function D: Heat Sensor

This is a detailed description of an individual function of our circuit. To access other functions and get an overview of the whole circuit, visit the Circuit page.

Introduction

Sensing the temperature is crucial for the lifecycle of many bacteria and viruses. [1] We adapt this function from a specific heat-inducible system and engineer it to fit our needs for activating the toxin release in CATE.

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. [2]

DNA thermosensors rely on the bending of DNA at lower temperatures, which enables cooperative binding of DNA-associated proteins (Figure 1). 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 heat sensor — Figure 1. DNA-based thermosensor.

RNA based thermosensors form a stem-loop in the messenger RNA, which hides the Shine-Dalgarno sequence and the AUG translation initiation codon (Figure 2). At higher temperatures, the hydrogen bonds of the stem loop break apart and the ribosomal subunits can associate with the RNA and initiate translation.

RNA thermosensors can act without a translated mediator protein. In contrast, for protein-based temperature sensing, a protein needs to be expressed and available in the cytoplasm (Figure 3). This comes with the potential to modulate the sensors characteristics by modulating the protein expression levels. This can be achieved by modifying the RBS sequence to generate small and easily screenable RBS libraries. [3] This is a reason why we use a protein based thermosensor.

Protein based heat sensor — Figure 3. 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. [4] The advantage of this system is the high on/off-ratio of up to 355 [4] 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 a promotor containing a binding site for TlpA called P_TlpA (Figure 4). 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. [4] [5]

TlpA heat sensor — Figure 4. CATE's protein-based thermosensor. Protein E is regulated by TlpA-mediated thermosensing in the circuit of CATE.

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. 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 physician decides to proceed with the treatment, he can use focused ultrasound, a technology already included in modern MRI devices. It can heat up patients tissue in a precisely defined area inside the body. Focused ultrasound is normally used with very high teperatures to destroy tumors. However, the strong heat diffuses out of the tumor and thus damages surrounding healthy tissue. To prevent this off-target damage, we use the same focused ultrasound technology at a lower temperature, just 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 (Figure 5). 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.

Release of azurin — Figure 5. The safety checkpoint 2. TlpA dimers bind P_TlpA and the accumulated Anti-Cancer Toxin (azurin) stays inside the cell. A pulse of focused ultrasound increases the temperature to 45 °C and unfolds the TlpA dimers. Unfolded TlpA does not bind the P_TlpA anymore. Unbound P_TlpA is a strong promoter and leads to protein E expression. Protein E interferes with cell wall synthesis and leads to Cell Lysis. Previously accumulated azurin is released to the extracellular space during lysis of CATE.

Following cell lysis, azurin is released to the extracellular space (Figure 5).

References