Figure 5.

Effects of LasR on production of virulence factors in P. aeruginosa isolates. (a) Complementation of a lasR mutant with the lasR restored proteolytic activity. Data represent the lysis zones on milk plate after 24 h of P. aeruginosa growth at 37 °C. (b) Analysis of LasB activity using ECR assays. Cell-free supernatant from bacterial culture grown for 18 hours were used. (c) Levels of pyocynain. Extraction of pyocyanin from the supernatant fraction of bacterial cultures grown for 48 hours were measured at 520 nm. Values of each experiment are presented as the mean ± SDs of tests performed in triplicate. Experiments were performed in three different occasions. ****P<0.0001; *P < 0.05. P values were obtained by one way ANOVA with Bonferroni test. (d) Immunoblotting analysis of secreted exotoxin A (ETA) and LasB. Equal amounts of proteins were loaded and subjected to SDS-PAGE. ETA and LasB were disclosed by anti-ETA antibody and anti-LasB antibody. Arrows show unknown small protein bands crossreacted with anti-ETA antibody. The ratios of ETA levels to LasB levels were shown underneath the blots. The blot of LasB and ExoA in strains PAO1, ΔlasR, and ΔlasR+were in the same gel. Strains bj13, bj13lasR, bj14 and bj14lasR were in the same gel and the full-length blots were shown in Supplemental Figure S5.