The Mechanism for Type I Interferon Induction by Mycobacterium tuberculosis is Bacterial Strain-Dependent (original) (raw)
Fig 4
Release of host, but not bacterial, DNA into the cytosol is mycobacterial strain-dependent and is associated with mitochondrial stress.
A-D) BMDM were infected with the indicated mycobacterial strains at an MOI of 5. 24 hr post infection, cells were collected and fractionated. Mitochondrial (A), nuclear (B), and bacterial (C,D) DNA in cytosolic fractions was quantified using gene-specific primers and normalized to the amount of each gene in lysates of unfractionated cells (shown as %). *p<0.05, **p<0.01 by one-way ANOVA with Tukey post-tests; means ± SD (n = 3). E) Mitochondrial proteins (CVα and PDH) in the organelle and cytosolic fractions were quantified on immunoblots. β-actin was used as the cytosolic loading control. Results are representative of 2 independent experiments. F) BMDM were cultured in the presence of galactose and absence of glucose for 24 hr and then infected with the indicated mycobacterial strains at MOI of 1, 5, and 10. 24 hr post infection luciferase and luciferin were added to cell lysates and ATP production was measured by luminescence. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Tukey post-tests for each MOI; means ± SD (n = 3). G-H) BMDM were infected with the indicated GFP-expressing mycobacterial strains at an MOI of 5. Live BMDM were stained with MitoSOX at 24 hr post infection and then fixed overnight. G) Representative images are shown, with Mtb shown in green and MitoSOX shown in red. H) 5 pictures were taken at 60x magnification for each chamber well and mean fluorescence intensity (MFI) of MitoSOX was determined for each infected BMDM using ImageJ. Results are representative of 3 independent experiments. *p<0.05, **p<0.01 by one-way ANOVA with Tukey post-tests; means ± SD (n = 3).