Expression of Biofilm-Degrading Enzymes in Plants and Automated High-Throughput Activity Screening Using Experimental Bacillus subtilis Biofilms.

P Opdensteinen,S J Dietz,J F Buyel,B B Gengenbach

doi:10.3389/fbioe.2021.708150

Abstract

Biofilm-forming bacteria are sources of infections because they are often resistant to antibiotics and chemical removal. Recombinant biofilm-degrading enzymes have the potential to remove biofilms gently, but they can be toxic toward microbial hosts and are therefore difficult to produce in bacteria. Here, we investigated Nicotiana species for the production of such enzymes using the dispersin B-like enzyme Lysobacter gummosus glyco 2 (Lg2) as a model. We first optimized transient Lg2 expression in plant cell packs using different subcellular targeting methods. We found that expression levels were transferable to differentiated plants, facilitating the scale-up of production. Our process yielded 20 mg kg−1 Lg2 in extracts but 0.3 mg kg−1 after purification, limited by losses during depth filtration. Next, we established an experimental biofilm assay to screen enzymes for degrading activity using different Bacillus subtilis strains. We then tested complex and chemically defined growth media for reproducible biofilm formation before converting the assay to an automated high-throughput screening format. Finally, we quantified the biofilm-degrading activity of Lg2 in comparison with commercial enzymes against our experimental biofilms, indicating that crude extracts can be screened directly. This ability will allow us to combine high-throughput expression in plant cell packs with automated activity screening.

Highlights

Many bacteria, including pathogens (Dufour et al, 2010), naturally form biofilms (Stiefel et al, 2016) to protect themselves against environmental threats such as UV light, disinfectants, antibiotics, and host immune effector mechanisms (Del Pozo, 2018)
We used our previously described automated high-throughput platform based on transient expression in plant cell packs (PCPs) prepared from tobacco Bright Yellow 2 (BY-2) cells (Gengenbach et al, 2020) to systematically investigate the expression of C-terminally His6-tagged Lysobacter gummosus glyco 2 (Lg2) in the cytosol, apoplast, ER, and chloroplasts, in each case using three different 5’ untranslated region (UTR) (Supplementary Table S1)
Lg2 accumulated to the highest levels in the cytosol and apoplast

Summary

Introduction

Many bacteria, including pathogens (Dufour et al, 2010), naturally form biofilms (Stiefel et al, 2016) to protect themselves against environmental threats such as UV light, disinfectants, antibiotics, and host immune effector mechanisms (Del Pozo, 2018). Harsh methods are unsuitable for the removal of biofilms formed by pathogens inside the Abbreviations: BY-2, Bright Yellow 2; EPS, exopolysaccharides; Lg2, Lysobacter gummosus glyco 2; PCPs, plant cell packs; PNAG, poly-N-acetyl-β-(1,6)-glucosamine; UTR, untranslated region. As an alternative to chemical and mechanical methods, enzymes can facilitate the dispersion of biofilms under mild conditions, including physiological temperatures (Stiefel et al, 2016). Such enzymes target the major components of biofilms, namely, exopolysaccharides (EPS), proteins, and nucleic acids (Roux et al, 2015). The efficiency of these enzymes varies depending on the composition of the biofilm, which in turn depends on the microorganisms, growth conditions, and environment (Roux et al, 2015)

Methods

Results

Conclusion