Nepetin reduces virulence factors expression by targeting ClpP against MRSA-induced pneumonia infection

Figures & data

Figure 1. Nepetin inhibits the activity of ClpP and does not affect the growth of bacteria. (a) Chemical structural of nepetin. (b) Determination of IC₅₀ values of nepetin on the fluorescent substrate Suc-LY-AMC.(c) The growth of USA300 is unaffected by 100 μM nepetin. Wild type USA300 was used as acontrol. 50 (d) Nepetin at 100 μM has no effect on the viability of Vero cells over 24 hours.

Figure 2. Nepetin binds to and inhibits ClpP activity. (a) the binding between nepetin and ClpP was examined by TSA. Nepetin caused a decrease in the thermal stability of ClpP. (b) western blot image and thermal shift assay curves showing that nepetin caused a decrease in the Tm of ClpP protein in the cell lysates of E. coli carrying pEt28a-clpP. Full western blot images are shown in Supplementary Fig. S3. (c) LSPR analysis demonstrates the kinetics of nepetin binding to ClpP. (d) Noncompetitive inhibition of nepetin on ClpP cleavage of Suc-LY-AMC. (e) Molecular docking predicted the binding pattern of nepetin and ClpP. (f) IC₅₀ assay of nepetin on WT-ClpP and two mutants Q47A-ClpP and S22A-ClpP. the two mutant proteins showed some degree of resistance to nepetin inhibition. Significance is calculated based on two-tailed t-test: **P < .01 and ***P < .001.

Figure 3. Expression of multiple virulence factors in S. aureus is inhibited by nepetin. (a) Expression levels of agr, RNAIII, hla, luks, psm-α and spa were determined by qPCR in the presence of 100 μM of nepetin. (b and c) Quantification of α-toxin and PVL expression levels in S. aureus USA300 and Newman under the effect of different concentrations of nepetin by western blot, and their corresponding gray value analysis. (d) The expression levels of nepetin on α-toxin in USA300 and Δclpp-USA300 were examined by Western blot. Nepetin lost its ability to inhibit α-toxin in Δclpp-USA300. (e and f) the effect of different concentrations of nepetin on the hemolytic capacity of S. aureus USA300 and USA300 supernatant. (g) Nepetin induces urease production, which causes the medium to show a pink color. Δclpp-USA300 was used as a positive control. Significance is calculated based on one-way ANOVA: **P < .01 and ***P < .001.

Figure 4. Nepetin protects mice from MRSA pneumonia. (a) Survival of mice treated with nepetin (100 mg/kg) at the indicated times after infection with USA300 (2e8 CFU/30 µl). Significance (p-value) in the panels except (a) is calculated using log-rank test: **P < .01 and ***P < .001. (b) Gross pathology and histopathology of S. aureus USA300 and USA300-Δclpp infected lung tissue from mice. Nepetin (100 mg/kg) treatment by subcutaneous injection. Scale bar, 50 μm. (c) the infectious bacterial load in the lung of mice with nepetin (100 mg/kg) treatment. in the graph, horizontal bars indicate the mean of bacterial load measurements, each dot represents a mouse. Significance is calculated based on one-way ANOVA: **P < .01 and ***P < .001.

Data availability statements

All data generated during this study are available on request from the corresponding authors.