AUTHOR=Su Tiantian , He Jing , Li Ningna , Liu Shiheng , Xu Sujuan , Gu Lichuan 

TITLE=A Rational Designed PslG With Normal Biofilm Hydrolysis and Enhanced Resistance to Trypsin-Like Protease Digestion

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.00760

DOI=10.3389/fmicb.2020.00760

ISSN=1664-302X

ABSTRACT=<p>A glycosyl hydrolase produced by <italic>Pseudomonas aeruginosa</italic>, PslG, has become a promising candidate for biofilm treatment because of its ability to inhibit and disperse biofilms by disrupting exopolysaccharide matrix at nanomolar concentrations. However, as a protein, PslG used for treatment may be degraded by the ubiquitous proteases (of which trypsin-like serine proteases are a major group) secreted by human cells. This would lead to an insufficient effective concentration of PslG. Here, based on the result of liquid chromatography–tandem mass spectrometry (LC-MS/MS) and structural analysis, we generate a PslG mutant (K286A/K433S) with greatly enhanced trypsin resistance. This measure raises IC<sub>50</sub> (the concentration of trypsin that can degrade 50% of protein in 30 min at 37°C) from 0.028 mg mL<sup>–1</sup> of the wild-type PslG to 0.283 mg mL<sup>–1</sup> of PslG<sup><italic>K</italic>286<italic>A/K</italic>433<italic>S</italic></sup>. In addition, biofilm inhibition assay shows that PslG<sup><italic>K</italic>286<italic>A</italic>/<italic>K</italic>433<italic>S</italic></sup> is much more efficient than wild-type PslG in the presence of trypsin. This indicates that PslG<sup><italic>K</italic>286<italic>A/K</italic>433<italic>S</italic></sup> is a better biofilm inhibitor than wild-type PslG in clinical use where trypsin-like proteases widely exist.</p>