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. 2023 Sep;10(25):e2302459.
doi: 10.1002/advs.202302459. Epub 2023 Jun 28.

Celastrol Combats Methicillin-Resistant Staphylococcus aureus by Targeting Δ1 -Pyrroline-5-Carboxylate Dehydrogenase

Zhongwei Yuan  1 , Jun Wang  1 , Qianwei Qu  1 , Zhenxin Zhu  1 , Marc Xu  2 , Mengmeng Zhao  1 , Chongxiang Sun  1 , Haixin Peng  1 , Xingyu Huang  1 , Yue Dong  1 , Chunliu Dong  1 , Yadan Zheng  1 , Shuguang Yuan  2 , Yanhua Li  1
Affiliations

Celastrol Combats Methicillin-Resistant Staphylococcus aureus by Targeting Δ1 -Pyrroline-5-Carboxylate Dehydrogenase

Zhongwei Yuan et al. Adv Sci (Weinh). 2023 Sep.

Abstract

The emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) raise a critical need for alternative therapeutic options. New antibacterial drugs and targets are required to combat MRSA-associated infections. Based on this study, celastrol, a natural product from the roots of Tripterygium wilfordii Hook. f., effectively combats MRSA in vitro and in vivo. Multi-omics analysis suggests that the molecular mechanism of action of celastrol may be related to Δ1 -pyrroline-5-carboxylate dehydrogenase (P5CDH). By comparing the properties of wild-type and rocA-deficient MRSA strains, it is demonstrated that P5CDH, the second enzyme of the proline catabolism pathway, is a tentative new target for antibacterial agents. Using molecular docking, bio-layer interferometry, and enzyme activity assays, it is confirmed that celastrol can affect the function of P5CDH. Furthermore, it is found through site-directed protein mutagenesis that the Lys205 and Glu208 residues are key for celastrol binding to P5CDH. Finally, mechanistic studies show that celastrol induces oxidative stress and inhibits DNA synthesis by binding to P5CDH. The findings of this study indicate that celastrol is a promising lead compound and validate P5CDH as a potential target for the development of novel drugs against MRSA.

Keywords: MRSA; P5CDH; celastrol; multi-omics; multiple pathways.

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Conflict of interest statement

Shuguang Yuan is the cofounder of AlphaMol Science Ltd.

Figures

Figure 1
Figure 1
Biosynthesis and degradation of Pro in bacteria.
Figure 2
Figure 2
Anti‐MRSA activity of celastrol in vitro. A) Chemical structure of celastrol. B) MIC of celastrol against MRSA USA300. PC: positive control (MH containing MRSA USA300 without celastrol); NC: negative control (MH containing celastrol without MRSA USA300); BC: blank control (MH only). C) MBC of celastrol against MRSA USA300. D) Growth curve of celastrol against MRSA USA300. E) Time‐kill curve of celastrol against MRSA USA300. F) Resistance development of MRSA USA300 to celastrol, vancomycin, and oxacillin. Values indicate fold changes (in log2) in MIC relative to the MIC of the first passage.
Figure 3
Figure 3
Anti‐MRSA activity of celastrol in vivo. A) Scheme of experimental protocol for G. mellonella infection, skin infection, and MRSA‐induced bacteremia models. Created with BioRender.com. B) Survival rates of G. mellonella larvae infected with MRSA USA300. Van: vancomycin, as a positive control; BC (blank control): only injected PBS without infections. C) Wound sizes in skin infection model after infection of MRSA USA300 in different groups. D) Bacterial burdens in wounds infected with MRSA USA300 in different groups. E) Image of wounds of mice infected with MRSA USA300 after 5 d of infection. E1: PBS treatment group; E2: low dose celastrol (0.1 mg kg−1) treatment group; E3: middle dose celastrol (0.2 mg kg−1) treatment group; E4: high dose celastrol (0.4 mg kg−1) treatment group; E5: vancomycin (0.4 mg kg−1) treatment group, as positive control. F) Histological evaluation of infected skin tissues of different groups. F1: PBS treatment group; F2: low dose celastrol (0.1 mg kg−1) treatment group; F3: middle dose celastrol (0.2 mg kg−1) treatment group; F4: high dose celastrol (0.4 mg kg−1) treatment group; and F5: vancomycin (0.4 mg kg−1) treatment group. G) Weight of kidneys of different groups. H) Histological evaluation of kidneys of different groups. H1: PBS treatment group; H2: low dose celastrol (6.25 mg kg−1) treatment group; H3: middle dose celastrol (12.5 mg kg−1) treatment group; H4: high dose celastrol (25 mg kg−1) treatment group; H5: linezolid (25 mg kg−1) treatment group, as a positive control. I) Bacterial burdens in the kidneys of different groups. NS, not significant; **p < 0.01 or *p < 0.05, compared with the PBS treatment group.
Figure 4
Figure 4
Multi‐omics analysis of MRSA USA300 treated with celastrol or DMSO. A) Scheme illustration of experimental protocol for multi‐omics. B) Violin plot. CV, coefficient of variation in different omics. C) KEGG enrichment analysis of multi‐omics pathways. D) P5CDH, as a potential anti‐MRSA target, was deduced from the multi‐omics analysis. Created with BioRender.com.
Figure 5
Figure 5
P5CDH is a potential antimicrobial target. A) Schematic model of construction of ΔrocA and Δ::rocA. B) PCR identification of ΔrocA and Δ::rocA. M: DL2000 DNA marker; 1: WT; 2: ΔrocA; 3: Δ::rocA. C) Growth curve of WT, ΔrocA, and Δ::rocA. D) MIC of celastrol against ΔrocA and Δ::rocA. PC: positive control (MH containing MRSA USA300 without celastrol); NC: negative control (MH containing celastrol without MRSA USA300); BC: blank control (MH only).
Figure 6
Figure 6
The site and binding affinity of celastrol to P5CDH. A) Homology model building of MRSA P5CDH (right) was performed according to the B. licheniformis P5CDH (left). B) Predicted binding mode of celastrol in P5CDH binding pocket obtained from molecular docking. C) Kinetic analysis by BLI of binding of celastrol to P5CDH. D) Cellular thermal shift assays to verify celastrol binding to P5CDH.
Figure 7
Figure 7
Key residues in the interaction between celastrol and P5CDH A) Position (red) of three residues (K205A, E208A, and D209A) in each protein. B) Structural models of three mutated proteins complexed with celastrol. C) Kinetic analysis by BLI of binding of celastrol to three mutated proteins. D) IC50 values of celastrol against P5CDH, K205A, E208A, and D209A. E) Normalized circular dichroism (CD) spectra of P5CDH in the presence and absence of celastrol.
Figure 8
Figure 8
Celastrol promotes oxidative damage and induces cell death. A) P5C content. B) Pro content. C) Ratio of NAD+/NADH. D) Intracellular ATP level. E) ROS level. F) Antioxidant capacity. G) Schematic diagram of celastrol inducing oxidative damage. Created with BioRender.com. WT: wild‐type strain group; ΔrocA: rocA knockout strain group; celastrol: WT treated with the celastrol group; Inhibitor: WT treated with glyoxylate group; Δ::rocA: rocA complemented strain group. NS, not significant; **p < 0.01 or *p < 0.05, compared with the WT group.
Figure 9
Figure 9
Detection of MRSA cell death under CLSM and SRFM. Bright field: cells were observed under bright field using CLSM; DAPI CLSM: cells stained with DAPI were observed by CLSM with excitation wavelength at 340 nm and emission wavelength at 488 nm; DAPI SRFM: cells stained with DAPI were observed by SRFM with excitation wavelength at 340 nm and emission wavelength at 488 nm; TUNEL CLSM: cells stained with TUNEL were observed by CLSM with excitation wavelength at 480 nm and emission wavelength at 520 nm. WT: wild‐type strain group; ΔrocA: rocA knockout strain group; celastrol: WT treated with celastrol group; Inhibitor: WT treated with glyoxylate group; Δ::rocA: rocA complemented strain group.
Figure 10
Figure 10
Celastrol inhibits DNA synthesis. A) Ratio of NADP+/NADPH. B) Glu content. C) Asp content. D) Fluorescence intensity of DNA. E) Protein content. F) SDS‐PAGE gel image. G) Schematic diagram of inhibition of synthesis of DNA and proteins by celastrol. Created with BioRender.com. M: maker; lanes 1: WT without treatment group; lanes 2: ΔrocA group; lanes 3: WT cells treated with celastrol group; lanes 4: WT cells treated with inhibitor group; lanes 5: Δ::rocA group. WT: wild‐type strain group; ΔrocA: rocA knockout strain group; celastrol: WT treated with celastrol group; Inhibitor: WT treated with glyoxylate group; Δ::rocA: rocA complemented strain group. NS, not significant; **p < 0.01 or *p < 0.05, compared with the WT group.
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