BACTerial Signal QUEnching of plant pathogens with Engineered enZymes – BACTSQUEEZ

The BactSqueez project used a combination of advanced methods to optimize enzymes capable of blocking quorum sensing in phytopathogenic bacteria.

- Bioinformatics analysis: Bioinformatics analyses of molecular dynamics were carried out to identify key areas of the SsoPox enzyme where mutations could enhance activity.

- Enzyme engineering: Targeted mutations were introduced into the SsoPox enzyme sequence. Micro-format libraries of several thousand enzymes were then produced.

- Activity screening: the enzyme libraries were screened using an efficient method specifically developed for this project, enabling the best candidate enzymes to be isolated.

- Biochemical & in vitro characterization: The lactonase activity of the best candidates is measured by kinetic assays. Then, the effectiveness of the variants in blocking the virulence of phytopathogens is studied in vitro.

-In planta tests: to test the effectiveness of enzymes in protecting plants, a dozen host-pathogen systems have been developed, at all stages of the plant's life.

The enzymes are then applied to these pathosystems to characterize plant protection against bacterial infection.

The first round of engineering has enabled us to isolate enzyme variants that are highly effective against target plant pathogens: variant G is 300 times more active than the wild-type enzyme on AHL C4-HSL, the main AHL used by the pathogen Serratia sp. 39006. The efficacy of this variant compared with the wild-type enzyme has been demonstrated in vitro. This enzyme significantly disrupts the bacterium's behavior and proteome, and reduces its virulence in a model of infection on vegetable slices infection model.

The IGY and KL variants are around 30 times more active than the wild-type enzyme on C6-HSL, AHL used by the pathogens D. solani and P. atrosepticum. The KL variant has demonstrated its efficacy on the pathogen P. atrosepticum. The enzyme reduces the production of proteins enabling plant infection, such as pectate lyases or cellulases, and reduces the bacterium's virulence in a model of tuber maceration and tomato seed germination.

The BactSqueez project offers promising applications in sustainable agriculture, thanks to enzymes developed to block quorum sensing in phytopathogenic bacteria.

These enzymes can serve as an ecological alternative to chemical treatments, enabling farmers to protect their crops without harming the environment. The catalog of enzymes created by the project will enable these solutions to be applied to a wide range of crops vulnerable to bacterial infection.

Furthermore, demonstrating the efficacy of these enzymes will pave the way for marketable formulations that can be integrated into current agricultural practices. Research will also be conducted to measure the biostimulation of plants by enzymes. All in all, the BactSqueez project has the potential to transform plant disease control methods, promoting more sustainable and responsible agriculture.

Phytopathogenic bacteria are responsible for various diseases in plants including canker, soft rot and fireblight. Considering the economic impact of these pathogens as well as the emergence of antimicrobial resistance and the toxicity of chemical biocides, developing sustainable and environmentally friendly antibacterial solutions is urgently needed. Disrupting bacterial quorum sensing (QS), a communication system commonly used by phytopathogenic bacteria to regulate infection, is highly promising. One partner of the project has developed a robust enzyme, referred to as SsoPox, targeting QS of many plant pathogens relying on acyl-homoserine lactones (AHL). This enzyme, also known as lactonase, was first shown to disrupt the QS of P. aeruginosa, an opportunistic pathogen with high antibioresistance ability. In the continuity of these preliminary results, BactSqueez will face a new challenge by extending the QQ approach to the degradation of AHL involved in the QS of highly problematic phytopathogens (Dickeya sp., Agrobacterium sp., Pectobacterium sp.). Thanks to a multidisciplinary consortium involving both academic and industrial partners, BactSqueez will combine computational design, enzyme engineering and plant microbiology to develop improved lactonases that will be biochemically characterized and evaluated at both in vitro and in planta levels. Involving an industrial partner, the enzymes generated will then be considered for concrete applications to limit the use of phytopharmaceutical chemicals thanks to an innovative and sustainable alternative. Moreover, enzymes have not been considered for agricultural purpose so far, the lactonase-based solutions will thus be highly disruptive and pave the way for further development in the field.