Fig 1.
Structures of GRA and its derivatives.
Table 1.
Primers used in this study.
Table 2.
MIC of glycyrrhetinic acid and its derivatives against S.aureus strains.
Fig 2.
Effect of GRA and GRA derivatives on S. aureus MW2 growth.
Overnight cultures of S. aureus MW2 were adjusted to an OD of 1.0. Then, 100 μl of bacterial culture was inoculated into 5 ml of TSB. The bacterial culture was incubated at 37°C with shaking. When the OD at 660 nm reached 0.3, various concentrations (■: control, ♦: 1/64 MIC, ▲: 1/16 MIC, ●: 1/4 MIC, ×: 1x MIC, +: 2x MIC) of GR-SU or GRA were added to the medium. The growth and colony counts were monitored. Three independent experiments were performed, and the mean ± SD was calculated. The data were analyzed for statistically significant differences compared to untreated control by a two-way ANOVA followed by Dunnett’s post hoc tests. *P<0.05.
Fig 3.
Effect of GRA and GRA derivatives on S. aureus cell viability under non-growing conditions.
Cell viability was evaluated under non-growing conditions following the addition of GRA or GR-SU. The methods are described in the Materials and Methods section. S. aureus MW2 was treated with 1x MIC and 2x MIC of GR-SU (grey bar) or GRA (white bar) in 10 mM sodium phosphate buffer. An untreated bacterial culture (black bar) was used as a control. After 1 and 2 h of incubation, the reaction mixtures (100 μl) were plated on trypticase soy agar and incubated at 37°C overnight. The colony forming units (CFUs) were determined as the total number of colonies identified on each plate. The antibacterial effect was calculated as the ratio of the number of surviving cells (survival rate %) to the total number of bacteria incubated. Three independent experiments were performed, and the mean ± SD was calculated. The data were analyzed for statistically significant differences compared to the untreated control by a two-way ANOVA followed by Dunnett’s post hoc test. Significant differences were not observed.
Table 3.
FICIs in combination of various antibiotics with GRA or GR-SU.
Fig 4.
Microarray analysis of genes with altered expression levels in response to GR-SU.
The microarray analysis was performed as described in the Materials and Methods section. The numbers of genes with 2-fold upregulated (grey bar) or downregulated (white bar) expression are indicated.
Fig 5.
Effects of GRA and GRA derivatives on the expression of RNAIII (a, b) and seh (c, d). Overnight cultures of S. aureus MW2 (108 cells) were inoculated into 5 ml of TSB and aerobically incubated at 37°C with shaking. When the OD at 660 nm reached 0.3, 1/4 MIC (a, c) or 1x MIC (b, d) of GR-SU or GRA was added to the culture (■: control, ♦: GR-SU, ▲: GRA). Bacterial cells were collected after 1, 2, 4 and 8 h of incubation. Then, RNA extraction and cDNA synthesis were performed. Quantitative PCR was performed using cDNA as the template DNA. Three independent experiments were performed and the mean ± SE was calculated.
Fig 6.
Effects of GRA and GRA derivatives on virulence factor expression.
Overnight cultures of S. aureus MW2 (108 cells) were inoculated into 5 ml of TSB and aerobically incubated at 37°C with shaking. When the OD at 660 nm reached 0.3, 1x MIC of GR-SU (a) or GRA (b) was added to the culture. The bacterial cells were collected after 2 h. Then, RNA extraction and cDNA synthesis were performed. A quantitative PCR was performed using cDNA as the template DNA. Three independent experiments were performed and the mean ± SE was calculated.
Table 4.
Effect of GRA and GR-SU on sugar and amino acids requirement.