Description

Introduction Antibyeotic sensor Antibyeotic deactivation Kill Switch References

Introduction

As we were brainstorming, trying to find a local problem that we would like to tackle, we decided that the One Health approach is in our field of interests. Tackling a problem which affects human health, animal health and environmental health seemed to be of utmost importance. We specifically targeted poultry farming because of our region (Epirus, where the city of Ioannina is located, has plenty of poultry farms) and the direct access to experts. Our goal is to reduce the antibiotics that accumulate in the environment from facilities like poultry farms. We chose two kinds of antibiotics, Tetracyclines and Macrolides, because they are used by both humans and animals and their mechanisms of action are well known.

Chicken manure has a remaining concentration of antibiotics (Xi et al., 2020) and this could lead to pressure added on the bacterial strains of this substrate to develop antibiotic resistance mechanisms. It is well-known that the soil near livestock facilities or treated with manure has higher concentrations of antibiotics (Zhao et al., 2017). Tetracyclines and macrolides are widely used in chicken manure (Cycoń et al., 2019), thus, leading to their accumulation in the substrate.

The basis of our project is the design of a synthetic bacterial strain that would reduce the antibiotic residues present in chicken manure after it is collected. Our engineered strain would have the necessary molecular mechanisms to uptake these antibiotics, deactivate them and when it has fulfilled its role be self-destructed to ensure the safety of its use. These roles will be accomplished through three genetic modules: Antibiotic Sensor – Antibiotic Deactivation – Kill Switch

Antibyeotic Sensor

In order to make this system dependent on the presence or absence of antibiotics, we inserted an antibiotic sensor in our design. This sensor contains two repressors of gene expression: TetR and MphR which can bind tetracyclines and macrolides respectively.

These two repressors of gene expression have the ability to bind to DNA depending on their recognition of antibiotics. Both regulatory mechanisms have been found naturally in bacterial strains in regulatory mechanisms underlying antibiotic resistance (Deng et al., 2013; Zheng et al., 2009).

In the absence of tetracyclines, TetR dimers bind to an operator that is specific for this repressor and is placed upstream of the promoter of a gene of interest. When tetracyclines are present, they can bind to TetR dimers and not allow their binding to the DNA operators. MphR is a protein with similar action as TetR, with the difference in the antibiotic binding ability (Cuthbertson & Nodwell, 2013). MphR can bind macrolides and depending on their presence or absence, regulate gene expression.

This regulatory module will sense the presence of antibiotics in the strains environment and regulate the survival of the bacterial strain.

Antibyeotic Deactivation

The biological role of our designed bacterial strain would be to inactivate tetracyclines and macrolides found in its near environment. This role can be accomplished with the expression of two antibiotic modifying enzymes, TetX2 and EreB that lead to the inactivation of tetracyclines and macrolides respectively. TetX2 is a tetracycline-inactivating enzyme and more specifically, a FAD-requiring monooxygenase, which inactivates different kinds of tetracyclines by regioselective hydroxylating them to 11a-hydroxytetracycline with NADPH as a cofactor and O2 as electron acceptor (Besharati et al., 2019; Yang et al., 2004). EreB is a macrolide esterase, which hydrolyses the macrolactone ring (Morar et al., 2012).

These two antibiotic deactivation enzymes will ensure that the bacterial strain, once applied in a substrate containing these two antibiotics, can lead to their inactivation and thus decontamination of the substrate from these two components.

Kill Switch

The third module of our design is a biosafety measure. If this bacterial strain was to be applied after future further research, it would have to be as safe as possible. For this reason, we decided to add a kill switch mechanism to the bacterial strain. This mechanism would be regulated by the antibiotic sensor. The two repressors (TetR, MphR) would regulate the expression of an antitoxin that would control the half-life of another protein, a toxin for the bacterial cell. This regulatory mechanism would allow the bacterial strain to be alive until it has modified and inactivated the amounts of tetracyclines and macrolides in its environment. When the concentration of the antibiotic is low enough to not be recognized by the repressors, the production of the antitoxin will stop enabling the expression of the toxin, leading our strain to self-destruction.

The toxin protein would be a protease, mf-lon, and the antitoxin would be a modified bpDNase1 that has the mf-lon recognition tag. This toxin-antitoxin system was originally designed by IISER-Tirupati and thus we decided to partner and test this system.

References