A Novel Insight into Dehydroleucodine Mediated Attenuation of Pseudomonas aeruginosa Virulence Mechanism

Figure 3

Effect of DhL on P. aeruginosa strain PAO1 protease activities. Staphylolytic LasA and elastolytic LasB activities were monitored in the absence or in the presence of DhL (0.12 mg/mL). (a) To measure the proteolytic activity of LasA within the supernatant fraction of the PAO1 growth culture, a staphylolytic assay was employed accounting the lysis of heat inactivated intact S. aureus at OD₅₉₅ as described in Section 2. Proteolytic activity of PAO1 supernatant (PAO1, □) was significantly (asterisk indicates ) inhibited when PAO1 were treated with DhL (PAO1 + DhL, ●). LB + S. aureus was used as control (○). Data represent the mean of three independent experiments ± SEM. (b) Levels of LasB elastolytic activity (within the PAO1 cultural supernatant with or without DhL treatment) were measured by Elastin Congo red assay at OD₄₉₅ as described in Section 2. PAO1 were grown in LB broth at 37°C for 16 hr. The cultures were adjusted to an OD₅₄₀ of 3.5–4.0 before harvesting to eliminate growth-related variations in elastolytic activity. Significant (asterisk indicated ) decrease in LasB activity was observed in PAO1 when treated with DhL. Data represent the mean of three independent experiments ± SEM. (c) Relative mRNA expression level of LasR gene at mid log phase from DhL treated P. aeruginosa shows fivefold inhibition compared to untreated population. Data represent the mean of three independent experiments ± SEM.

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