Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant clinical challenge due to its multidrug resistance, particularly to β-lactam antibiotics. This study comprehensively evaluated the natural compound emodin for its anti-MRSA activity, mechanisms of action, and potential for synergy with β-lactam antibiotics. Our findings demonstrate that emodin dose-dependently inhibits MRSA growth and abrogates biofilm formation at 2× MIC. Mechanistic studies revealed that emodin compromises cell membrane and wall integrity, induces oxidative stress, and downregulates the virulence factors SPA and EsxA. Furthermore, emodin acted synergistically with β-lactam antibiotics: it enhanced the ability of cefalexin to block bacterial adhesion and invasion of HaCat cells, and potentiated the efficacy of amoxicillin in clearing MRSA from infected macrophages. In conclusion, emodin employs a multi-target mechanism against MRSA and can resensitize the bacterium to conventional β-lactam antibiotics, presenting a promising strategy for combination therapy that may help curb antibiotic use and resistance development.

Oleum cinnamomi (OCM) is a volatile component of the Cinnamomum cassia Presl in the Lauraceae family, which displays broad-spectrum antibacterial properties. It has been found that OCM has a significant inhibitory effect against Cutibacterium acnes (C. acnes), but the precise target and molecular mechanism are still not fully understood. In this study, the antibacterial activity of OCM against C. acnes and its potential effect on cell membranes were elucidated. Metabolomics methods were used to reveal metabolic pathways, and proteomics was used to explore the targets of OCM inhibiting C. acnes. The yield of the OCM was 3.3% (w/w). A total of 19 compounds were identified, representing 96.213% of the total OCM composition, with the major constituents being phenylpropanoids (36.84%), sesquiterpenoids (26.32%), and monoterpenoids (15.79%). The main component identified was trans-cinnamaldehyde (85.308%). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of OCM on C. acnes were 60 µg/mL and 180 µg/mL, respectively. The modified proteomics results indicate that cinnamaldehyde was the main bioactive ingredient within OCM, which covalently modifies the ABC transporter adenosine triphosphate (ATP)-binding protein and nicotinamide adenine dinucleotide (NADH)-quinone oxidoreductase, hindering the amino acid transport process, and disrupting the balance between NADH and nicotinamide adenine dinucleoside phosphorus (NAD⁺), thereby hindering energy metabolism. We have reported for the first time that OCM exerts an antibacterial effect by covalent binding of cinnamaldehyde to target proteins, providing potential and interesting targets to explore new control strategies for gram-positive anaerobic bacteria.

Introduction: Oleum Cinnamomi (OC) is a volatile oil extracted by steam distillation from the dried branches and leaves of Cinnamomum cassia Presl, a plant belonging to the Lauraceae family. For centuries, OC has been utilized as a food preservative and flavoring agent, demonstrating potent inhibitory effects against bacteria and fungi. It is particularly effective in controlling infections caused by Methicillin-Resistant Staphylococcus epidermidis (MRSE), which often parasitizes the skin surface. To uncover the target and molecular mechanism by which OC eradicates MRSE, this study initially assessed the impact of OC and its primary constituents on oxidative stress in MRSE cells. Methods: Mass spectrometry was employed to identify the target and covalent binding sites of OC, while a kit was used to monitor changes in key biomolecules of MRSE cells exposed to OC. Additionally, the efficacy of OC in inhibiting MRSE adhesion and infection of RAW 264.7 mouse macrophages was evaluated. Results: The findings revealed that OC's main components, cinnamaldehyde and 2-methoxycinnamaldehyde, covalently modify MRSE and AhpC. This modification disrupts the AhpC-AhpE regeneration cycle, thereby disturbing both enzymatic and non-enzymatic redox homeostasis. It leads to intracellular ROS accumulation and effectively prevents MRSE from adhering to RAW 264.7 mouse macrophages. In response to ROS detoxification, MRSE attempts to upregulate the expression of TCA cycle-related proteins. However, the continuous accumulation of ROS inactivates the [Fe-S] protein of Aconase (ACO), hindering ACO's catalytic conversion of citric acid to isocitrate. This results in sustained intracellular accumulation of citric acid, limiting the TCA cycle and ATP generation. Simultaneously, enzymes involved in reduction catalysis, such as superoxide dismutase (SOD), peroxidase reductase (Prx), and glutathione synthase (GCL), are collectively inactivated. OC induces oxidative stress in MRSE, depleting GSH and triggering lipid peroxidation, which in turn induces MRSE to undergo ferroptosis. Discussion: This covalent inhibition strategy targeting AhpC to induce ferroptosis offers a promising approach for effectively treating and preventing MRSE infections, thereby opening new avenues for combating drug-resistant pathogen infections.

Background: Preventing Cutibacterium acnes ( C. acnes ) adhesion to human skin is essential for acne control, yet the molecular determinants of adhesion-associated drug resistance remain undefined and no pharmacological agents specifically blocking C. acnes attachment are available. Objective: To evaluate oleum cinnamomi (OC), a natural cinnamon oil, as a potential anti-adhesive modality against C. acnes . Methods: In vitro antibacterial assays, quantitative chemoproteomics, and keratinocyte infection models were integrated to quantify OC-mediated inhibition of C. acnes adhesion and biofilm formation, map covalent targets of OC electrophiles cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), and delineate downstream metabolic perturbations via untargeted metabolomics and redox profiling. Results: OC, CA and MCA concentration-dependently reduced C. acnes adhesion (> 80%) without rapid bactericidal activity. Both CA and MCA covalently modified Cys359 of enolase (ENO), suppressing glycolytic flux and ATP generation. Independently, CA formed a thiohemiacetal adduct with coenzyme A (CoA), blocking acyl-transfer reactions required for fatty-acid metabolism and membrane biogenesis. The combined effects elevated the NADH/NAD ⁺ ratio, disrupted oligosaccharide homeostasis, and impaired biofilm exopolysaccharide biosynthesis with altered monosaccharide composition, thereby compromising biofilm formation and driving C. acnes into a metabolically compromised state. Conclusion: OC-derived electrophiles act as metabolic modulators that incapacitate C. acnes adhesion and biofilm development through orthogonal inhibition of ENO and CoA-dependent pathways, providing a mechanistic blueprint for potential topical anti-acne therapies that circumvent conventional antibiotic resistance.