Marine-Fungi-Derived Gliotoxin Promotes Autophagy to Suppress Mycobacteria tuberculosis Infection in Macrophage

Abstract

The Mycobacterium tuberculosis (MTB) infection causes tuberculosis (TB) and has been a long-standing public-health threat. It is urgent that we discover novel antitubercular agents to manage the increased incidence of multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains of MTB and tackle the adverse effects of the first- and second-line antitubercular drugs. We previously found that gliotoxin (1), 12, 13-dihydroxy-fumitremorgin C (2), and helvolic acid (3) from the cultures of a deep-sea-derived fungus, Aspergillus sp. SCSIO Ind09F01, showed direct anti-TB effects. As macrophages represent the first line of the host defense system against a mycobacteria infection, here we showed that the gliotoxin exerted potent anti-tuberculosis effects in human THP-1-derived macrophages and mouse-macrophage-leukemia cell line RAW 264.7, using CFU assay and laser confocal scanning microscope analysis. Mechanistically, gliotoxin apparently increased the ratio of LC3-II/LC3-I and Atg5 expression, but did not influence macrophage polarization, IL-1β, TNF-a, IL-10 production upon MTB infection, or ROS generation. Further study revealed that 3-MA could suppress gliotoxin-promoted autophagy and restore gliotoxin-inhibited MTB infection, indicating that gliotoxin-inhibited MTB infection can be treated through autophagy in macrophages. Therefore, we propose that marine fungi-derived gliotoxin holds the promise for the development of novel drugs for TB therapy.

Keywords: Mycobacterium tuberculosis (MTB); autophagy; gliotoxin; macrophages; marine natural product.

Figure 1

Gliotoxin inhibited MTB infection in THP-1-derived macrophages. (A–C) Chemical structures of marine-fungi-derived gliotoxin (1), 12, 13-dihydroxy-fumitremorgin (2), and helvolic acid (3) were shown and CCK8 kit was used to detect cytotoxicity of THP-1-derived macrophages with treatment of different concentrations of these compounds for 48 h. (D) CCK8 assay was applied to detect cell proliferation of THP-1-derived macrophages with treatment of different concentrations of isoniazid for 48 h. (E,F) CFU assay has been determined for MTB survival with treatments of 0.25 μM gliotoxin, 10 μM 12, 13-dihydroxy-fumitremorgin C, 25 μM helvolic acid, and 10 μM isoniazid in THP-1-derived macrophages upon 0 h and 48 h of MTB infection. (Data presented as mean ± SD; n = 4, at least three independent experiments with four replicates each; * p ≤ 0.05 was considered as statistically significant; ns, not significant).

Figure 2

Gliotoxin suppressed MTB infection in RAW 264.7. (A) CCK8 assay showed the cytotoxicity of different concentrations of gliotoxin to RAW264.7 cells for 48 h. (B) Treating RAW264.7 cells with 0.25 μM gliotoxin, the intracellular mycobacterial viability was determined using CFU assay at 24 h and 48 h post infection. (C,D) RAW264.7 cells were treated with 0.25 μM gliotoxin, 1 μM isoniazid or the combination, CFU assay, and bacteria were stained with Texas red inside macrophages, with laser confocal microscopy assay applied. (Data presented as mean ± SD; n = 4, at least three independent experiments with four replicates each; * p ≤ 0.05 was considered as statistically significant; ns, not significant).

Figure 3

Gliotoxin promoted autophagy upon MTB infection. (A) M1 macrophage surface markers CD80, CD86, MHC-II, and M2 macrophage surface markers CD163, CD206 expression using flow cytometry analysis and (B) cytokines of IL-6, IL-1β, TNF-α, and IL-10 mRNA and protein expression were detected using qRT-PCR, Western blot, or ELISA analysis with gliotoxin treatment for 24 h in MTB-infected RAW264.7 cells. β-actin served as an internal reference. (C) ROS production was determined using an ROS Activity Assay Kit, and (D,E) ratio of LC3-II/LC3-I and Beclin1, Atg5, Atg7 expression were detected using Western blot assay with gliotoxin treatment for 24 h in MTB-infected RAW264.7 cells and THP-1 macrophages. β-actin served as an internal reference. (Data presented as mean ± SD; n = 3–4; ** p ≤ 0.01 was considered as statistically significant; ns, not significant.).

Figure 4

Gliotoxin inhibited MTB infection by promoting autophagy machinery. (A) Ratio of LC3-II/LC3-I was detected using Western blot assay with gliotoxin treatment or 3-MA treatment upon 24 h of MTB infection in RAW264.7 cells. β-actin served as an internal reference. (B) The intracellular bacterial load was determined using CFU assay and (C) laser confocal microscopy assay with gliotoxin, 3-MA or their combination at 48 h post infection. (D) Ratio of LC3-II/LC3-I was detected using Western blot assay with gliotoxin treatment or 3-MA treatment upon 24 h of MTB infection in THP-1 macrophages. β-actin served as an internal reference. (E) Texas red-stained bacteria in THP-1 macrophages with gliotoxin, 3-MA or their combination at 24 h post infection were detected using laser confocal microscopy assay. (Data presented as mean ± SD; n = 4; at least three independent experiments each with four replicates; * p ≤ 0.05; ** p ≤ 0.01 were considered as statistically significant).