Development of a synthetic sensor system for the detection of infectious and inflammatory signals

Bacterial infections are a major challenge, e.g. upon implant transplantation. Especially the formation of biofilms protects the bacteria from effective treatment. Here, anti-infective treatments needs to be performed early with onset of infection. Inspired by synthetic biological devices this study aimed at engineering a cellular device which can detect infection or inflammation derived signals and transmit these signals to synthetic cassettes. Activation of the type 1 interferon (IFN) system was chosen as a physiological hallmark for viral and bacterial infections as well as a marker for inflammation. To rewire infection related input signals to synthetic cassettes the synthetic doxycycline dependent transactivator gene (tTA) was integrated into the endogenous murine Mx2 chromosomal locus. This locus has been assigned to be specifically induced by type I IFN. Integration of a single copy was performed using CRISPR/Cas9 assisted homologous recombination in murine embryonic stem (mES) cells. Subsequently the IFN signalling was coupled to luciferase expression by integration of a transactivator dependent expression cassette. The engineered ES cells displayed an attenuated IFN response as a consequence of endogenous regulatory mechanisms governing in stem cells. Upon differentiation these cells displayed high induction levels of reporter expression which could be reduced to basal levels by addition of doxycycline even with ongoing IFN signalling being present. Introduction of an autoregulatory circuit could increase the sensitivity without losing the ability of downregulation reporter expression in presence of doxycycline. In an alternative approach the tTA transactivator was integrated into a bacterial artificial chromosome (BAC) vector compromising about 150kb of the cellular Mx2 locus. Stable integration of the modified BAC vector into NIH/3T3 cells displayed a higher sensitivity and an overall higher luciferase expression compared to the single copy as obtained with the CRISPR/Cas9 approach. These systems could detect pathophysiological IFN concentrations in vivo and still preserved their ability to be regulated in presence of doxycycline. Finally, rewiring of infection signals to expression of Il10, an anti-inflammatory cytokine, was achieved. Together, this study represents the proof of concept for the functional rewiring of infectious signals to reporter and anti-inflammatory cues and offers great potential with regard to translational approaches.