Molecular mechanism underlying Oleum Cinnamomi-induced ferroptosis in MRSE via covalent modification of AhpC

Abstract

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.

Keywords: AhpC; MRSE; Oleum Cinnamomi (OC); ROS; covalent inhibitors; metabolic pathways.

FIGURE 1

The experimental flowchart in this study. MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; ROS, reactive oxygen species; DEPs, differentially expressed proteins; PPI, protein–protein interaction; PFK-1, phosphofructokinase-1; ICDHs, isocitrate dehydrogenases; ACO, aconitase; NAD-MDH, NAD-dependent malate dehydrogenase; SOD, superoxide dismutase; Prx, peroxiredoxin; GCL, glutamate-cysteine ligase; Prx-Px, peroxiredoxin-peroxidase; GSH, glutathione; LPO, lipid peroxidation; NADH/NAD⁺, nicotinamide adenine dinucleotide.

FIGURE 2

Typical total ion chromatograms of Oleum Cinnamomi analyzed by GC-MS.

FIGURE 3

OC induces ROS generation on MRSE. During the logarithmic growth phase, MRSE was exposed to 1×MIC and 2×MIC concentrations of OC for a duration of 4 h, with 0.1% DMSO serving as the control. Intracellular ROS were detected using the CM-H₂DCFDA probe, and flow cytometry was employed to monitor and generate fluorescence histograms (A1). The medium fluorescence intensity was utilized for quantification (A2). The same method was applied to evaluate CA-induced ROS generation in MRSE, resulting in the generation of fluorescence histograms (B1), with medium fluorescence intensity again used for quantification (B2). Likewise, MCA-induced ROS generation in MRSE was assessed using the same approach, leading to the creation of fluorescence histograms (C1), with medium fluorescence intensity employed for quantification (C2).

FIGURE 4

The effect of OC-treated MRSE on autophagy in RAW264.7 macrophages. (A) MRSE was treated with OC at 0 and 1×MIC for 4 h, followed by infection with CFDA-SE. RAW264.7 cells were subsequently exposed to MRSE at a multiplicity of infection (MOI) of 10:1 for 2 h. The adhesion of MRSE to RAW264.7 cells was observed using a fluorescence microscope with the green fluorescence channel for CFDA-SE. (B) A histogram of the fluorescence intensity obtained from flow cytometry was generated. (C) The number of viable MRSE cells within RAW264.7 cells and the percentage of RAW264.7 cells were recorded on Mueller-Hinton agar plates after incubation with OC-treated MRSE and RAW264.7 cells.

FIGURE 5

Detection of AhpC binding epitopes covalently modified by OC through modification proteomics. (A) Amino acid sequence of the retrieved protein Q5HRY1. (B) Mass-to-charge (m/z) values of the main active components of OC: Cinnamaldehyde (CA) and Methoxycinnamaldehyde (MCA). (C) Detected representative peptide segments and their modification sites. (D1, D2) represents the primary mass spectrum of QHPGEVcPAK (CA Modification at Cysteine Residue C7) peptide segment, and (E1, E2) represents peptide consensus view of b, y-ions of the secondary mass spectrum of peptide segment QHPGEVcPAK (MCA Modification at Cysteine Residue C7]).

FIGURE 6

AhpC-Protein interactions analysis for DEPs. (A) PCA analysis of the control group and the 1×MIC group. (B) Volcano plot analysis of differentially expressed proteins obtained from control and OC-treated samples. (C) Analysis of AhpC-protein interactions, with the intensity of the data indicated by line thickness; thicker lines signify a higher confidence in the interaction. Each node in the network represents a protein, while each edge denotes a protein-protein association, with hidden disconnected nodes included in the network. (D) Fold change (FC) of dehydrogenases among the differentially expressed proteins.

FIGURE 7

OC disrupts MRSE oxidative stress defense, inducing ferroptosis. MRSE was treated with a specific concentration of OC for 4 h, followed by centrifugation and washing with PBS. After extraction according to the kit instructions, the concentration of bacterial protein was determined using PCA. (A) Expression of intracellular ROS detoxification-related reductases, (B) Measurement of intracellular superoxide dismutase (SOD) and peroxiredoxin (Prx) activity, (C) Expression of glutathione (GSH)-related enzymes, (D) Measurement of intracellular GCL and glutathione peroxidase (GSH-Px) activity, (E) Expression of iron ion transport-related proteins, (F) Measurement of intracellular GSH content, (G) Measurement of lipid peroxidation content.

FIGURE 8

Regulation of Glycolysis and the TCA Cycle in MRSE to Counter OC-Mediated Oxidative Stress. Exposure of MRSE to 1×MIC of OC: (A) Expression levels of glycolysis-related enzymes in MRSE, (B) Expression levels of TCA cycle-related enzymes in MRSE. Exposure of MRSE to 1×MIC and 2×MIC of OC: (C) Activity of the PFK-1 enzyme and intracellular levels of NADH, NAD⁺, and ATP in MRSE, (D) Activity of NAD-MDH and ICDHs enzymes in MRSE cells, (E) Activity of the ACO enzyme and citrate content in MRSE cells.

FIGURE 9

Protein interaction analysis focused on SERP1169. (A) SERP1169-protein interaction analysis: The strength of data support is indicated by the thickness of the lines, with thicker lines representing greater confidence in the interactions. Each node in the network represents a protein, while each edge signifies protein-protein associations. (B) Significant Differential Proteins in the SERP1169- protein interactions analysis. (C) Expression of key fatty acid enzymes.

FIGURE 10

OC-covalent modification of AhpC induces ROS and ferroptosis in MRSE: mechanistic diagram.