It is one of our main goals to engineer CATE such that the release of payload is controlled on different levels. The first level is the preferential accumulation of bacteria in solid tumors upon intravenous administration. According to literature, after 4 days E. Coli Nissle is exclusively found in tumor tissues. Up to this point however, small numbers of the bacteria have been found also in liver and spleen [1]. Therefore, the second level of control is the bacteria themselves – they should be able to autonomously decide if they have reached tumor tissue. Only then level 3 control, thermoactivation by focused ultrasound (LINK), comes into play.

Interpreting the Environment

To realize the second level of control, namely avoid activation of our engineered system outside of tumors, we devised a strategy for CATE to be able to differentiate between healthy and tumor tissue. This is achieved by AND-logic integration of two inputs: AHL and Lactate. AHL is produced by the bacteria themselves and reaches high concentrations only in case the population is dense – ergo only in tumors. The second input is lactate, a chemical produced in high amounts by cancer cells as a consequence of defects in cellular respiration and other aberrations, even under normoxic conditions [2]. Hence, a combination of lactate and AHL is a unique molecular pattern that should only occur in tumor tissue.

Mechanism of Action

In order to achieve such AND-gate behaviour, we relied on the previously characterized parts LldP/LldR and Plldr as well as Plux and LuxR . LldP is a transmembrane protein that imports L-lactate. It is thought that LldR binds to two sites on the DNA, termed O1 and O2, and leads to formation of a DNA-loop that “hides” the region in between O1 and O2 from the transcription complex. Upon binding of L-lactate to LldR it undergoes a conformational change that leads to opening of the loop whereby intervening regions are exposed again [3]. Conversely, LuxR dimerizes upon binding to N-Acyl homoserine lactone (AHL). The resulting complex then binds to the Plux promoter and recruits RNA polymerase [4].

We reasoned that flanking the Plux promoter with O1 and O2 should lead to AND-gate behaviour. In the absence of Lactate, Plux should be looped and therefore inaccessible to LuxR, independent of AHL being present or not. If AHL is present but lactate is not, Plux is exposed but will not be activated. Only in the case where both inducers are present, no loop is formed and Plux is accessible to dimerized LuxR. See the illustration for a graphical representation of the action of our synthetic AND-gate promoter.