FIGURE 6.

In vivo effects of phendione-based compounds on the lasB-induced toxicity against G. mellonella larvae. Firstly, the toxic effects of cell-free culture supernatants obtained from ATCC 27853 and 09HC were evaluated. In this way, fresh and thermally inactivated (100°C/5 min) bacterial supernatants (100 μg) were injected into the larvae hemocoel (10 per group), and the larvae survivability was followed up for 7 consecutively days (A). The presence of lasB in the pseudomonal supernatants was confirmed by the cleavage of the specific fluorogenic peptide substrate (aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide) and by gelatin-zymography. The molecular mass on the right evidences the elastase proteolytic band. Note the significant difference regarding the elastase production by both P. aeruginosa strains (B). Subsequently, Cu-phendione was tested on its ability to revert the toxic effects of both bacterial supernatants (C) and purified lasB molecules (D). In this set of experiments, different concentrations (1, 10, and 100 μg of proteins) of P. aeruginosa ATCC 27853 secretions and purified lasB molecules (1 μg) were treated or not with 50 μM Cu-phendione for 30 min at room temperature. Then, both untreated and Cu-phendione-neutralized systems were injected into larvae hemocoel (10 per group), and the larvae survival was checked daily for 7 days. The survival data were plotted using the Kaplan–Meier method using GraphPad Prism v 7.0.