Tamarixetin Attenuated the Virulence of <i>Staphylococcus aureus</i> by Directly Targeting Caseinolytic Protease P.
Song W, Wang B, Sui L, Shi Y, Ren X, Wang X, Kong X, Hou J, Wang L, Wei L, Luan Y, Guan J, Zhao Y.
<i>Staphylococcus aureus</i>, especially drug-resistant <i>S. aureus</i> infections, is a worldwide healthcare challenge. There is a growing focus on antivirulence therapy against <i>S. aureus</i>. Caseinolytic protease p (ClpP) is a protein hydrolase essential for pathogenicity in <i>S. aureus</i>. A flavonoid compound, tamarixetin, which was screened in this work, was specifically able to inhibit the hydrolytic activity of ClpP on the fluorescent substrate Suc-LY-AMC with an IC<sub>50</sub> of 49.73 μM, without affecting the growth of methicillin-resistant <i>S. aureus</i> strain USA300 and was without obvious cytotoxicity. Further assays found that tamarixetin inhibited the transcription of <i>hla</i>, <i>agr</i>, <i>RNAIII</i>, <i>pvl</i>, <i>PSM-α</i>, and <i>spa</i> genes as well as suppressed the protein expression levels of Hla and PVL. Moreover, tamarixetin was observed to dramatically inhibit the hemolytic activity of <i>hla</i> in <i>S. aureus</i>. Consistent with that of <i>S. aureus</i> USA300-Δ<i>clpP</i>, tamarixetin was shown to increase urease expression. The thermal shift and cellular thermal shift assays showed that tamarixetin markedly changed the thermal stability of ClpP. The dissociation constant (<i>K</i><sub>D</sub>) value of tamarixetin with ClpP was 2.52 × 10<sup>-6</sup> M measured by surface plasmon resonance. The molecular docking and ClpP point mutation results also demonstrated that tamarixetin had a strong interaction with ClpP. <i>In vivo</i> study showed that tamarixetin was effective in protecting mice from <i>S. aureus</i> pneumonia by increasing survival, reducing lung tissue load, and slowing down the infiltration of inflammatory factors. In addition, tamarixetin was able to enhance the antibacterial activity of cefotaxime in combination. In conclusion, tamarixetin was promising as a ClpP inhibitor for <i>S. aureus</i> infections.