Chemical structure of Thiostrepton (TST).

In vitro activity of TST. The activity of TST against Mab abscessus CIP 104,536 S morphotype in comparison with CLA and CFX in cation-adjusted Mueller–Hinton medium.

TST-mediated regulatory effects on cytokine production in bone-marrow-derived macrophage (BMDMs) during 104,536 S infection. BMDMs were infected with Mab CIP 104,536 S morphotype and treated with TST for 6 h; then, they were subjected to quantitative real-time Polymerase Chain Reaction (PCR) analysis for measurement of the mRNA expression of TNF-α (A), IL-1β (B), IL-12p40 (C), IL-6 (D), and IL-10 (E), and protein expression of TNF-α (F). * P < 0.05, ** P < 0.01, *** P < 0.001, compared with Mab-infected cells. Data are representative of three independent experiments, and values represent mean ± standard error (SE) from three independent experiments performed in triplicate. TST, thiostrepton; CLA, clarithromycin.

TST-mediated immunomodulatory effects on lipopolysaccharide (LPS)-induced cytokine expression in BMDMs. BMDMs were stimulated with LPS (100 ng/mL) in the presence or absence of TST (2 μM) for 6 h; then, they were subjected to quantitative real-time PCR analysis for measurement of the mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C), and IL-10 (D). *** P < 0.001, compared with LPS-stimulated cells. Data are representative of three independent experiments, and values represent mean ± standard error (SE) from three independent experiments performed in triplicate. TST, thiostrepton.

Intracellular activity of TST. The activity of TST on intracellular Mab CIP 104,536 S was examined in THP-1 cells. Data represent mean ± standard error (SE) from three independent experiments. ** P < 0.01; *** P < 0.001, when comparing untreated samples with samples treated with TST. UT, untreated; TST, thiostrepton; CLA, clarithromycin.

Evaluation of in vivo TST activity on Mab CIP 104536 R morphotype expressing mWasabi infection. (A) Mab expressing mWasabi (≈400 CFU) was infected via caudal vein infection and visualized in untreated or drug-treated embryos. (B) Bacterial loads of untreated, TST, and CLA treated embryos. Results are expressed as mean log₁₀ CFU per embryo from three independent experiments. Significant difference (*** P < 0.0001) compared with untreated control. Survival of Mab- infected embryos treated at 1 and 5 μM of TST (C) and CLA (D) in comparison with untreated infected embryos and non-treated control (n = 20, representative of three independent experiments). Survival curves were compared with Log-rank (Mantel-Cox) test (* P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant). TST, thiostrepton; CLA, clarithromycin.