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. 2022 Jul 25:13:963901.
doi: 10.3389/fmicb.2022.963901. eCollection 2022.

The Assessment on Synergistic Activity of Ebselen and Silver Ion Against Yersinia pseudotuberculosis

Chuanjiang Dong  1 , Wei Chen  2   3 , Lili Zou  1   2   3 , Binbin Liu  1   2   3 , Kaihong Deng  1   2   3 , Dingrui Guo  2   3 , Peng Wang  1 , Hao Chen  4 , Helen Wang  5 , Jun Wang  6
Affiliations

The Assessment on Synergistic Activity of Ebselen and Silver Ion Against Yersinia pseudotuberculosis

Chuanjiang Dong et al. Front Microbiol. .

Abstract

Yersinia pseudotuberculosis is a foodborne zoonotic bacterium that is pathogenic to guinea pigs, rabbits, and mice. It also causes pseudotuberculosis in humans. However, it still lacked the scientific basis for control. Here, we found out that Ebselen (EbSe) exhibited synergistic antibacterial activity with silver nitrate (Ag+) against Y. pseudotuberculosis YpIII strain with high efficacy in vitro using UV-visible light absorption spectrum, 5,5'-dithiobis-(2-nitrobenzoic acid), laser scanning confocal microscope, flow cytometry, transmission electron microscopy and Western blotting assays. The depletion of total glutathione (GSH) amount and inhibition of thioredoxin reductase (TrxR) activity in thiol-dependent redox system revealed the destructiveness of EbSe-Ag+-caused intracellular oxidative stress. Furthermore, a YpIII-caused mice gastroenteritis model was constructed. EbSe-Ag+ significantly reduced bacterial loads with low toxicity. It also down-regulated the expression levels of interferon (IL)-1β and tumor necrosis factor-α, up-regulated the expression level of IL-10 on-site. All the in vivo results demonstrated the antibacterial activity and immune-modulatory property of EbSe-Ag+. Collectively, these results provided academic fundament for further analysis and development of EbSe-Ag+ as the antibacterial agents for pseudotuberculosis control.

Keywords: Ebselen; Yersinia pseudotuberculosis; antibacterial activity; immune-modulatory property; silver nitrate.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
EbSe-Ag+ shows synergistic antibacterial activity against YpIII. YpIII-GFP and YpIII-mCherry were cultured to A600 of 0.4, diluted 1:1,000 times, and treated with EbSe-Ag+. (A,C) At 16 h; (B,D) at 24 h; (A,B) YpIII-GFP; (C,D) YpIII-mCherry.
FIGURE 2
FIGURE 2
EbSe-Ag+ has a bactericidal effect against YpIII. YpIII-GFP were cultured to A600 of 0.4, diluted 1:1,000 times, and treated with either EbSe, Ag+ or EbSe-Ag+. (A) LSCM results showed EbSe-Ag+ has a bactericidal effect against YPIII-GFP; (B) quantitative values.
FIGURE 3
FIGURE 3
EbSe-Ag+ influences YpIII morphology. YpIII-GFP were cultured to A600 of 0.4 and treated with EbSe-Ag+. TEM results showed EbSe-Ag+ influences YPIII-GFP morphology.
FIGURE 4
FIGURE 4
The PAE effect of EbSe-Ag+ against YpIII. YpIII-GFP and YpIII-mCherry were cultured to A600 of 0.4, diluted 1:1,000 times, and treated with EbSe-Ag+. The results showed EbSe-Ag+ has a PAE of 4 h. (A,C) 8 μM EbSe and a serial concentration of Ag+; (B,D) 8 μM EbSe and 0.5 μM Ag+; (A,B) YpIII-GFP; (C,D) YpIII-mCherry.
FIGURE 5
FIGURE 5
DTT protects YpIII from EbSe-Ag+. YpIII-GFP and YpIII-mCherry were cultured to A600 of 0.4, diluted 1:1,000 times, and treated with DTT followed with EbSe-Ag+. (A,D) At 16 h; (B,E) at 20 h; (C,F) at 24 h; (A–C) YpIII-GFP; (B–E) YpIII-mCherry. (G) YpIII-GFP was cultured to 0.4 and treated with EbSe-Ag+ with DTT, and the morphological changes were observed by TEM.
FIGURE 6
FIGURE 6
EbSe-Ag+ upregulates ROS production in YpIII. YpIII-mCherry was cultured to A600 of 0.4 and treated with EbSe-Ag+. (A) Cells were stained with 5 μM CellROX™ Deep Red Reagent for 30 min at 37°C and detected by FCM; (B) quantitative values.
FIGURE 7
FIGURE 7
EbSe-Ag+ inhibits TrxR activity and depletes GSH in YpIII. YpIII-GFP and YpIII-mCherry were cultured to A600 of 0.4 and treated with EbSe-Ag+. DTNB assay was used to detect the TrxR activity and GSH amount. (A,B) At 30 min; (C,D) 0–5 min slope; (A,C) YpIII-GFP; (B,D) YpIII-mCherry.
FIGURE 8
FIGURE 8
Effect of EbSe-Ag+ on Trx1 and S-PSSG expressions in YpIII. YpIII-GPF and YpIII-mCherry were cultured to A600 of 0.4 and treated with EbSe-Ag+. WB assay was used to detect (A,C) the Trx1 expression level and (E,G) S-PSSG expression level; (B,D) quantitative values of the Trx1 expression level; (F,H) quantitative values of the S-PSSG expression level; (A,B,E,F) YpIII-GFP; (C,D,G,H) YpIII-mCherry.
FIGURE 9
FIGURE 9
EbSe-Ag+ protects mice from YpIII-caused pseudotuberculosis. YpIII-illuminous were orally administrated into mice, and further treated with EbSe-Ag+. (A,B) Mice were monitored for bioluminescent emission using IVIS lumina II (Caliper LifeSciences) at 1-, 4-, and 7-days post-infection. (C,D) Mice were euthanized, the gastrointestinal tracts were removed, and imaged by bioluminescent imaging to analyze bacterial localization within gastrointestinal tracts at 1-, 4-, and 7-days post-infection. IC: infection (illuminous) control.
FIGURE 10
FIGURE 10
EbSe-Ag+ has immune-modulatory property. YpIII-illuminous were orally administrated into mice, and further treated with EbSe-Ag+. Mice intestinal tracts were rolled, fixed, and embedded. Paraffin sections were used to perform H&E stain and detect cytokine (IL-1β, TNF-α and IL-10) levels by IHC analysis.
FIGURE 11
FIGURE 11
Routine blood analysis of EbSe-Ag+-treated mice. YpIII-illuminous were orally administrated into mice and further treated with EbSe-Ag+. Mice blood was collected 7 days post-infection, and the numbers of (A) WBC and (B) neutrophils, the amounts of (C) ALT, (D) AST, (E) Urea, and (F) creatine were detected.
FIGURE 12
FIGURE 12
The illustration of bactericidal effect of EbSe-Ag+.
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