Topical Therapeutic Efficacy of Ebselen Against Multidrug-Resistant Staphylococcus aureus LT-1 Targeting Thioredoxin Reductase

Abstract

As a thiol-dependent enzyme, thioredoxin reductase (TrxR) is a promising antibacterial drug target. Ebselen, an organo-selenium with well-characterized toxicology and pharmacology, was recently reported to have potent antibacterial activity against Staphylococcus aureus. In this paper, we demonstrated that ebselen has strong bactericidal activity against multidrug-resistant (MDR) S. aureus based on taking TrxR as a major target and disruption of the redox microenvironment. Further, the topical therapeutic efficacy of ebselen for staphylococcal skin infections was assessed in a rat model. Treatment with ebselen significantly reduced the bacterial load and the expression of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β) in S. aureus skin lesions; further, wound healing and pathological changes were obvious improved in ebselen-treated rats compare to controls. Finally, ebselen was found to sensitize S. aureus to curcumin, which may be due to their synergistic effects in inhibiting bacterial TrxR. Altogether, ebselen is an effective topical antibacterial agent in animal model of MDR S. aureus LT-1 skin infection. This may lay the foundation for further analysis and development of ebselen as an antibacterial agent for topical treatment of MDR staphylococcal infections.

Keywords: Staphylococcus aureus; curcumin; ebselen; thioredoxin reductase; topical treatment.

FIGURE 1

Chemical structure of ebselen.

FIGURE 2

Antibacterial effect of ebselen on Staphylococcus aureus through targeting bacterial TrxR. S. aureus LT-1 cells grown to DO 600 nm of 0.4 and diluted 100 times were treated with serial dilution of ebselen, and gentamycin was used as positive control. (A) Antibacterial effect of ebselen on the growth of S. aureus. Bacterial growth was presented by measuring OD_{600 nm}. S. aureus LT-1 cells grown to DO 600 nm of 0.4 and were treated with 22 μg/ml ebselen. (B) Mean ± SD of propidium iodide (PI)-stained S. aureus LT-1 by Flow cytometry; (C–H) Transmission electron microscopy of S. aureus treated with ebselen; (C,D) control; (E,F) 22 μg/ml ebselen; (G,H) 64 μg/ml gentamycin; (C,E,G) 15000x; (D,F,H) 25000x; (I) TrxR activity was assayed for DTNB reduction in the presence of Trx in S. aureus LT-1 extracts; (J) Mean fluorescent intensity (MFI) Means ±SD of H₂DCF-DA-stained S. aureus LT-1 were detected to present ROS level. (^∗∗P < 0.01; ^∗∗∗P < 0.001; student’s t-test. Data are presented as means ±SD of three independent experiments).

FIGURE 3

Therapeutic efficacy of ebselen in treating staphylococcal skin lesions. Rats were treated with 25 mg/Kg ebselen, 5 mg/Kg gentamycin (positive control) and PBS once daily for 5 days, respectively. (A) The bacterial load was calculated by counting the colonies; (B) The wound healing was evaluated according to the formula as follows: F = (A-B)/A^∗100%, where F: wound healing rate, A: original wound area, B: wound area post-treatment. (^∗P < 0.05; ^∗∗P < 0.01; student’s t-test. Data are presented as means ±SD of three independent experiments).

FIGURE 4

Efficacy of ebselen on cytokines production in staphylococcal skin lesions. Blood serum from each rat was used for cytokine detection by ELISA. (A) Tumor necrosis factor-α (TNF-α); (B) interleukin-6 (IL-6); (C) Interleukin-1 beta (IL-1β). (^∗P < 0.05; ^∗∗P < 0.01; student’s t-test. Data are presented as means ±SD of three independent experiments).

FIGURE 5

Histological results of ebselen-treated staphylococcal skin lesions. The new formative tissues from staphylococcal skin lesions rats with different treatments were used for pathological detection. (A–D) HE staining, red arrow indicated granulation tissues, purple arrow indicated fibroblasts; (E–H) Masson staining, blue color indicated collagenous fibers; (I–L) IHC using CD64, brown dots indicated CD64 positive cells; (M–P) IHC using S100a4, brown dots indicated S100a4 positive cells; (A,E,I,M) Control; (B,F,J,N) Ebselen; (C,G,K,O) Gentamycin; (D,H,L,P) PBS.

FIGURE 6

Synergistic antibacterial effect of ebselen and curcumin on the growth of S. aureus. S. aureus ATCC25923 grown to DO 600 nm of 0.4 and diluted 100 times were treated with serial concentrations of ebselen and curcumin in combination. (A,B) Antibacterial effect of ebselen and curcumin on the growth of S. aureus, and cell viability was presented by measuring OD_{600 nm}, (A) Ebselen and curcumin were added at the same time in the very beginning; (B) Curcumin was added at the very beginning, and ebselen was added 60 min later. S. aureus ATCC25923 grown to DO 600 nm of 0.4 were treated with 5 μM ebselen and 80 μM curcumin in combination. (C) Mean ±SD of propidium iodide (PI)-stained S. aureus by Flow cytometry. (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; student’s t-test. Data are presented as means ±SD of three independent experiments).

FIGURE 7

The inhibition activity of TrxR in ebselen and curcumin-treated S. aureus. S. aureus ATCC25923 grown to DO 600 nm of 0.4 were treated with 5 μM ebselen and 80 μM curcumin in combination. (A,B) TrxR activity was assayed using DTNB reduction in the presence of Trx in S. aureus extracts; (A) Slope curve; (B) Means ±SD; (C) Mean fluorescent intensity (MFI) of Means ± SD of H₂DCF-DA-stained S. aureus were detected to present ROS level. (^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; student’s t-test. Data are presented as means ±SD of three independent experiments).