Journal: Journal of Innate Immunity

Article Title: Inhibition of Pseudomonas aeruginosa Biofilm Formation by Peptidyl-Arginine Deiminases 2 and 4

doi: 10.1159/000550256

Figure Lengend Snippet: PAD2 and PAD4 affect PGO2330 QS gene expression and production of QS-related virulence factors. PGO2330 (5 × 10 5 CFU/mL) was cultured with rPAD2 or rPAD4 (20 n m ) for 24 h. Following incubation, cells were collected for qPCR analysis and the cell-free media (CFM) was assessed for virulence factor production. a qPCR analysis of lasR , lasI , rhlR , rhlI , and mvfR mRNA expression (normalised to ProC ) of rPAD2- and rPAD4-treated PGO2330 cells, relative to the control. b eDNA release, protease production, rhamnolipid release, and pyocyanin production of rPAD2- and rPAD4-treated PGO2330 cells, relative to the control. Data were analysed by a one-way ANOVA followed by multiple comparison Tukey’s post hoc test and presented as mean ± SEM; n = 5 biological replicates. c IL-6 and IL-8 gene expression in HBE cells 3 and 24 h post-incubation with rPAD2, rPAD4 (20 n m ), or P. aeruginosa (PAO1) LPS (10 µg/mL) (positive control). Gene expression was assessed by qPCR and calculated relative to untreated control ( n = 3 biological replicates). d Inhibition of PGO2330 protease activity present in 24 h CFM by protease inhibitor (ProtInh) cocktail. Data were analysed by a paired t test and presented as mean ± SEM; n = 3 biological replicates. e Western blot analysis of rPAD2 or rPAD4 (20 n m ) exposed to 24 h growth PGO2330 CFM for 2 h. f Densitometric analysis of PGO2330 CFM-treated rPADs relative to untreated rPADs (con). Data were analysed by a one-way ANOVA followed by multiple comparison Tukey’s post hoc test and presented as mean ± SEM; n = 5 biological replicates. SEM, standard error of the mean.

Article Snippet: Protease inhibitor (ProtInh) cocktail (ThermoFisher) was used as negative control.

Techniques: Gene Expression, Cell Culture, Incubation, Expressing, Control, Comparison, Positive Control, Inhibition, Activity Assay, Protease Inhibitor, Western Blot