Autophagy induction targeting mTORC1 enhances Mycobacterium tuberculosis replication in HIV co-infected human macrophages - PubMed (original) (raw)
Autophagy induction targeting mTORC1 enhances Mycobacterium tuberculosis replication in HIV co-infected human macrophages
Anna-Maria Andersson et al. Sci Rep. 2016.
Abstract
To survive and replicate in macrophages Mycobacterium tuberculosis (Mtb) has developed strategies to subvert host defence mechanisms, including autophagy. Autophagy induction has the potential to clear Mtb, but little is known about its effect during controlled tuberculosis and HIV co-infection. Mammalian target of rapamycin complex1 (mTORC1) inhibitors were used to induce autophagy in human macrophages pre-infected with HIV-1BaL and infected with a low dose of Mtb (co-infected), or single Mtb infected (single infected). The controlled Mtb infection was disrupted upon mTOR inhibition resulting in increased Mtb replication in a dose-dependent manner which was more pronounced during co-infection. The increased Mtb replication could be explained by the marked reduction in phagosome acidification upon mTOR inhibition. Autophagy stimulation targeting mTORC1 clearly induced a basal autophagy with flux that was unlinked to the subcellular environment of the Mtb vacuoles, which showed a concurrent suppression in acidification and maturation/flux. Overall our findings indicate that mTOR inhibition during Mtb or HIV/Mtb co-infection interferes with phagosomal maturation, thereby supporting mycobacterial growth during low-dose and controlled infection. Therefore pharmacological induction of autophagy through targeting of the canonical mTORC1-pathway should be handled with caution during controlled tuberculosis, since this could have serious consequences for patients with HIV/Mtb co-infection.
Figures
Figure 1. HIV/Mtb co-infection increases LC3B puncta formation and association of LC3B to Mtb phagosomes.
(a) Representative micrographs of LC3B puncta formation (arrowheads) and co-localization to Mtb phagosomes (arrows) in hMDMs infected with HIV (7 days) and/or H37Ra (MOI = 1, 2 h). Blue: DAPI, green: Mtb, red: LC3B (AlexaFluor594). (b) Percentage of LC3B positive hMDMs (3 puncta or more). (c) Percentage of LC3B positive Mtb phagosomes. (d) Percentage of hMDMs infected with the indicated number of bacteria/cell, showing no difference in Mtb infection with or without HIV. Data are mean ± SEM from 10 independent experiments in which 50–100 phagosomes were counted for each condition. *p < 0.05 using paired Student t-test.
Figure 2. mTOR inhibition using rapamycin accelerates H37Ra/H37Rv replication in both single and HIV co-infected hMDMs.
(a) hMDMs were pre-infected with/without HIV for three days before infection with H37Ra or H37Rv (MOI = 1) for 2 hours. Rapamycin (rapa; 1 μM) or 3-MA (1 mM) was added for 1, 3, and 7 days and the signal from luciferase expressing H37Ra or H37Rv in cell lysates and supernatant were measured. “Total bacteria” is the combined supernatant + intracellular pool of bacteria. (b) At the indicated time-points hMDMs (MQ) viability compared to uninfected hMDMs was measured using calcein AM uptake. Data are mean ± SEM from 6 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using paired Student t-test.
Figure 3. The efficient mTORC1 inhibitor Torin1 causes a dose-dependent increase in Mtb growth in co-infected hMDMs at low MOI.
(a) Representative immunoblots from two independent experiments showing the dose-response of Torin1 and rapamycin on the autophagy markers LC3B and SQSTM1 (p62) and phosphorylation of the mTORC1 downstream targets S6 and 4EBP1, with their respective β-actin loading controls, after 6 h treatment. Full length of the cropped blots are shown in Supplementary Fig. S3. (b) hMDMs were infected at the indicated MOI for 2 h, and then treated with/without 250 nM Torin1 for 3 days. The signal from luciferase expressing H37Rv was quantified, and the graph shows the ratio in total bacteria (lysate + supernatants) for Torin1 treated vs. untreated. Data are mean ± SEM from 6 independent experiments. *p < 0.05 using paired Student t-test. (c) hMDMs were pre-infected with/without HIV for seven days before infected with H37Ra (MOI = 0.2) for 2 hours. hMDMs were then incubated with/without rapamycin or Torin1 at increasing concentrations for 3 days. Graphs show the level of intracellular bacteria in cell lysates compared to day 0. Data are mean ± SEM from triplicate, representative of two independent experiments.
Figure 4. Same effect of early and late autophagy induction in Mtb infected hMDMs.
hMDMs were pre-infected with/without HIV for seven days before infected with H37Rv (MOI = 1) for 2 hours. hMDMs were then incubated with/without Torin1 (250 nM) for 3 days, added either directly or 3 days post infection. The graph shows the level of bacteria in cell lysates and supernatants. Data are mean ± SEM from 5 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 were * represent the significance for lysate while (*) represents the significance for total bacteria (lysate + supernatant) using paired Student t-test. (See also Supplementary Fig. S2 for CFU confirmation of these lysates).
Figure 5. HIV/Mtb co-infection inhibits phagosomal fusion with lysosomes, which is further decreased upon autophagy induction.
(a) Representative micrographs of LysoTracker Deep Red (LTDR) co-localization to phagosomes in hMDMs infected with yeast (MOI = 5) or co-infected with HIV/H37Rv for 6 h (7 days pre-infection with HIV), unstimulated and stimulated with Torin1 (250 nM) the last 4 h. The arrows in the HIV/H37Rv co-infected micrographs indicate co-localization to some of the Mtb phagosomes. All yeast particles in the lower micrographs exhibited co-localization. Green: Mtb or yeast, red: LTDR, yellow: co-localization. (b) Percentage of LTDR co-localization with yeast or H37Ra (MOI = 1) phagosomes 6 h post infection. (c) Percentage of LTDR co-localization with H37Rv (MOI = 1) phagosomes 6 and 24 h post infection. (d) hMDMs were pretreated with bafilomycin (baf; 100 nM) for 1h before infection with luciferase-expressing H37Rv (MOI = 1) for 2 h. Extracellular bacteria were washed away, and baf was re-added every 12 h. The combined luminescence signal from supernatant and hMDM-lysate (=total bacteria) are shown. Data are mean ± SEM with *p < 0.05 and **p < 0.01 using paired Student t-test, of 3 independent experiment for (d) and six independent experiments for LysoTracker data at 6 h and eight independent experiments for 24 h (n = 6 for yeast) in which 100–200 phagosomes were counted for each condition.
Figure 6. Mtb inhibits autophagic flux, and autophagy induction cause further build-up of SQSTM1 in Mtb phagosomes.
(a) Representative micrographs of SQSTM1 recruitment/accumulation to phagosomes in hMDMs infected with yeast (MOI = 5) or co-infected with HIV and Mtb (MOI = 1) for 6 h (7 days pre-infection with HIV), unstimulated and stimulated with Torin1 (250 nM) the last 4 h. Some cells were treated with bafilomycin (baf; 100 nM) 1 h prior to Mtb/yeast infection. The arrows indicate SQSTM1 co-localization to Mtb or yeast phagosomes. Green: Mtb or yeast, red: SQSTM1. (b) Percentage of SQSTM1 co-localization with yeast or H37Ra phagosomes. Data are mean ± SEM with **p < 0.01 using paired Student t-test, of six independent experiments (n = 3 for yeast+baf) in which 100–300 phagosomes were counted for each condition.
Figure 7. Torin1-induced autophagy and flux is cellular and not localized to Mtb phagosomes
. (a) Representative immunoblots from seven independent experiments showing the autophagy markers LC3B and SQSTM1 (p62) and the phosphorylation of the mTORC1 downstream targets S6 and 4EBP1, with their respective β-actin loading controls. The hMDMs were pre-infected for seven days with HIV before 6 h Mtb infection (MOI = 1), with the addition of Torin1 (250 nM) the last 4 h. Full length of the cropped blots are shown in Supplementary Fig. S6. (b–f) Densitometry measurements normalized to their respective β-actin control and presented as ratio over control without Torin1, shown as mean ± SEM with *p < 0.05 and **p < 0.01 using repeated measurement ANOVA comparing all treatments against its control (n = 7).
Figure 8. HIV modulated expression of essential ATG genes during co-infection.
hMDMs were pre-infected with/without HIV for seven days before 6 h Mtb infection (MOI = 1), adding Torin1 (250 nM) the last 4 h. Trizol was added to the infected and stimulated hMDMs to extract RNA. RNA was extracted from the same samples as analyzed for protein expression in Fig. 7. The gene expression were analyzed for: (a) SQSTM1, (b) LC3B, (c) Beclin1, (d) ATG4A, (e) ATG5, (f) ATG12, and (g) ATG16L2. The changes in gene expression are presented as ratios over control without Torin1, shown as mean ± SEM with *p < 0.05 and **p < 0.01 using repeated measures ANOVA (n = 5–6).
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