Phloretin is a natural chalcone with antibacterial and anti-inflammatory effects. This study investigated the anti-acne activity of phloretin against Propionibacterium acnes-induced skin infection and the potential target proteins of its anti-inflammatory and antibacterial effects. Phloretin potently inhibited the growth of P. acnes and P. acnes-induced Toll-like receptor (TLR) 2-mediated inflammatory signaling in human keratinocytes. Secreted embryonic alkaline phosphatase assay confirmed that the anti-inflammatory activity of phloretin is associated with the P. acnes-stimulated TLR2-mediated NF-κB signaling pathway. Phloretin significantly decreased the level of phosphorylated c-Jun N-terminal kinase (JNK), showing a binding affinity of 1.184 × 10−5 M−1. We also found that phloretin binds with micromolar affinity to P. acnes β-ketoacyl acyl carrier protein (ACP) synthase III (KAS III), an enzyme involved in fatty acid synthesis. Conformation-sensitive native polyacrylamide gel electrophoresis showed that phloretin reduced KAS III-mediated 3-ketoacyl ACP production by over 66%. A docking study revealed that phloretin interacts with the active sites of JNK1 and KAS III, suggesting their involvement in P. acnes-induced inflammation and their potential as targets for the antibacterial activity of phloretin. These results demonstrate that phloretin may be useful in the prevention or treatment of P. acnes infection.
To survive in diverse environments, bacteria adapt by changing the composition of their cell membrane fatty acids. Compared with aerobic bacteria, Cutibacterium acnes has much greater contents of branched-chain fatty acids (BCFAs) in the cell membrane, which helps it survive in anaerobic environments. To synthesize BCFAs, C. acnes acyl carrier protein (CaACP) has to transfer growing branched acyl intermediates from its hydrophobic cavity to fatty acid synthases. CaACP contains an unconserved, distinctive Cys50 in its hydrophobic pocket, which corresponds to Leu in other bacterial acyl carrier proteins (ACPs). Herein, we investigated the substrate specificity of CaACP and the importance of Cys50 in its structural stability. We mutated Cys50 to Leu (C50L mutant) and measured the melting temperatures (Tms) of both CaACP and the C50L mutant by performing circular dichroism experiments. The Tm of CaACP was very low (49.6 °C), whereas that of C50L mutant was 55.5 °C. Hydrogen/deuterium exchange experiments revealed that wild-type CaACP showed extremely fast exchange rates within 50 min, whereas amide peaks of the C50L mutant in the heteronuclear single quantum coherence spectrum remained up to 200 min, thereby implying that Cys50 is the key residue contributing to the structural stability of CaACP. We also monitored chemical shift perturbations upon apo to holo, apo to butyryl, and apo to isobutyryl conversion, confirming that CaACP can accommodate isobutyryl BCFAs. These results provide a preliminary understanding into the substrate specificity of CaACPs for the production of BCFAs necessary to maintain cell membrane fluidity under anaerobic environments.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.