Tripterhyponoid A from Tripterygium hypoglaucum Inhibiting MRSA by Multiple Mechanisms

Abstract

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) and its biofilm-forming ability underscore the limitations of current antibiotics. In this study, a new compound named tripterhyponoid A was found to effectively combat MRSA, with an MIC of 2.0 μg/mL. It inhibited biofilm formation by downregulating genes related to the quorum sensing (QS) pathway (sarA, agrA, agrB, agrC, agrD, and hld) and eradicated mature biofilms. Furthermore, it induced DNA damage by binding to bacterial DNA, enhancing its efficiency against MRSA. Therefore, its anti-MRSA properties with multiple mechanisms of action make it less prone to developing resistance over 20 days. In addition, it reduced the bacterial load and regulated the levels of inflammatory cytokines IL-6 and IL-10 at the wound site in a mouse skin infection model. This paper provides the first in-depth investigation of the mechanisms of triterpenoids against MRSA by inhibiting the expression of QS system genes and binding to DNA.

Keywords: DNA binding; anti-MRSA; antibiofilm; quorum sensing; tripterhyponoid A.

Figure 1

Structure elucidation of tripterhyponoid A. (A) Chemical structure. (B) Key correlations of ¹H-¹H COSY, HMBC, and ROESY spectra. (C) Experimental and calculated CD spectra.

Figure 2

Antibacterial activity of tripterhyponoid A in vitro. (A,B) Growth curves against MRSA003 and USA300. (C,D) Time-kill curves against MRSA003 and USA300. (E,F) Development of resistance in MRSA003 and VRE. Values indicating fold changes in MIC relative to the MIC of the initial passage. TA, tripterhyponoid A; VAN, vancomycin hydrochloride.

Figure 3

Transcriptomics analysis of USA300 treated by tripterhyponoid A. (A) Principal component analysis. (B) The distribution of differentially expressed genes (DEGs) in a volcano plot, with red dots representing upregulated genes and blue dots representing downregulated genes. (C,D) KEGG enrichment analysis for downregulated and upregulated genes, respectively. Con, DMSO; TA, tripterhyponoid A.

Figure 4

Antibiofilm mechanism of tripterhyponoid A (TA) against MRSA. (A) Model of agr-QS system inhibition mechanism (by FigDraw). The green arrows indicate genes downregulated by TA. (B) The RT-qPCR analysis of genes involved in the QS system. Values are mean ± SD (n = 3). *** p < 0.001 vs. CON.

Figure 5

The antibiofilm activity of tripterhyponoid A. (A) The inhibitory effect on a MRSA biofilm. (B) The elimination effect on a mature MRSA biofilm. (C) Fluorescence images of protein and nucleic acid staining for MRSA biofilm disruption. Scale bar: 100 μm. (D) The 3D fluorescence confocal micrographs of a USA300 biofilm matrix stained with FilmTracer™ SYPRO^® Ruby Biofilm Matrix Stain. Scale bar: 20 μm. Values are mean ± SD (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. CON. CON, DMSO; TA, tripterhyponoid A; VAN, vancomycin hydrochloride.

Figure 6

Pathway analysis of DEGs related to DNA replication and repair by tripterhyponoid A (by FigDraw). (A) DNA replication pathway. (B) DNA damage response pathway. (C) Folate biosynthesis and sulfur relay system pathways. Among them, the red text indicates that the DEGs were upregulated.

Figure 7

Tripterhyponoid A binds to USA300 genomic DNA. (A) Gel retardation analysis. Bands 1–5: tripterhyponoid A/DNA weight ratios of 200/1, 100/1, 50/1, 25/1, and 25/8, respectively; Band 6: untreated DNA, Band 7: marker. (B) UV spectra of interaction between tripterhyponoid A and DNA (50 ng/μL). (C) CD spectra of DNA in the presence of tripterhyponoid A. (D) The complex structure of tripterhyponoid A and DNA (left) and a detailed view of the binding bonds (middle, right).

Figure 8

Anti-MRSA activity of tripterhyponoid A (TA) in mouse skin infection model. (A) Experimental protocol. (B) Representative photographs of wounds infected with MRSA003 after 7 d of treatment. (C) Wound sizes after infection with MRSA003 in different groups. (D) Bacterial burdens in wounds on the 7th day post-infection. (E) Quantification of IL-6 and IL-10 in skin tissue. Model: PBS treatment group; VAN, vancomycin; CTX, cyclophosphamide. ### p < 0.001, compared with CK; *** p < 0.001, ** p < 0.01, or * p < 0.05, compared with model group. (F) Histological evaluation of mouse skin tissue at different magnifications. Skin tissue necrosis (black arrow), subcutaneous bleeding (grey arrow), granulocyte and lymphocyte infiltration (purple arrow), epidermal thickening (blue arrow), dermis bleeding (brown arrow), granulation tissue and fibrous connective tissue proliferation (orange arrow), new blood vessels (red arrow). Original magnification ×40 or ×200.