COVID-19, the biggest pandemic over the past century, has caused tremendous damage to global society. With SARS-CoV-2 spreading all over the world, another parallel public health issues should be taken into consideration: nosocomial infections. Among nosocomial infections, Ventilator-Associated Pneumonia (VAP) was a severe health problem caused by inhaling pathogen bacteria during ventilation, which subsequently contributed to high-density biofilm formation in respiratory tract and even led to death.

Having consulted the medical staff and researchers in the frontline against coronavirus, we found VAP caused by gram-negative pathogen bacteria pervasive and tricky to deal with. Considering the massive use of ventilators during the epidemic and potential hazards, we’re determined to develop a synthetic biology therapeutic approach against ventilator-associated pneumonia.

The interaction between pathogen bacteria and hosts is considered an arms race, in which immune cells act as ‘police squads’, pathogen bacteria act as ‘criminals’ and their quorum sensing systems as ‘communication tools’. Inspired by the movie ‘The Negotiator’, we proposed ‘negotiator’ probiotics with rationally designed genetic modules targeted at gram-negative quorum sensing systems could take down the battleground.

As a proof-of-concept of our project, we chose the opportunistic pathogen bacteria Pseudomonas aeruginosa as the ‘criminals’, and engineered our ‘negotiator’ probiotics, Escherichia coli Nissle 1917, with two modules: Quenching Module and Sensing Module. In Quenching Module, quorum quenching enzymes were heterologously overexpressed to parley with ‘criminals’ by degrading common autoinducers-acyl homoserine lactones (AHLs). In Sensing Module, the characteristic autoinducer-Pseudomonas Quinolone Signal (PQS) was recognized to guide the precise excretion of chemokines for recruiting ‘police squads’.

In addition, to provide ‘the negotiator’ with an evolving arsenal, we’re reconstituting quorum sensing pathways in E. coli lysate-based cell free expression systems for rapid and high-throughput screening of novel quorum sensing inhibitors. Aimed at antimicrobial resistance crisis, we anticipated these in vitro systems highly applicable to achieve sustainable development goals.

Most importantly, to supplement the wet-lab work, interconnected multidisciplinary methods were harnessed to demonstrate our project from diverse perspectives (see model and hardware pages). In addition, we scheduled thorough routes for ‘the negotiator’ to advance from theory to practice, in which the education goals were fulfilled, and ultimately to a commercially feasible product (see human practices and real-world applications).