Extended Data Fig. 9: The persistence phenotype of the rv1339 STB-K mutant (KC9A1) is due in part to enhanced resistance to NO. | Nature Microbiology

Extended Data Fig. 9: The persistence phenotype of the rv1339 STB-K mutant (KC9A1) is due in part to enhanced resistance to NO.

From: Parallel in vivo experimental evolution reveals that increased stress resistance was key for the emergence of persistent tuberculosis bacilli

Extended Data Fig. 9

NOS2-/- mice or C57BL/6 wild-type controls (WT) were infected via aerosol with approximately 100 c.f.u. of STB-K or KC9A1 and bacterial burden in the a) lung and b) spleen was quantified at 56 days post infection by c.f.u. plating on solid medium. Results are shown as the log10 c.f.u. for individual mice and the mean. n = 5 individual mice for each time point and each strain (except for KC9A1 which had n = 6 NOS2-/- individual mice) over one experiment. When bacteria were undetectable, the c.f.u. value was set at the limit of detection (log10 2; indicated by the broken horizontal line) for the purposes of statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with the Bonferroni post-hoc test. a, STB-K (WT) vs KC9A1 (WT); P = 0.0015, STB-K (WT) vs KC9A1 (NOS2-/-); P < 0.0001, KC9A1 (WT) vs STB-K (NOS2-/-); P = 0.0037; STB-K (NOS2-/-) vs KC9A1 (NOS2-/-); P = 0.0001, b) STB-K (WT) vs KC9A1 (WT); P = 0.0005, STB-K (WT) vs KC9A1 (NOS2-/-); P < 0.0001, KC9A1 (WT) vs STB-K (NOS2-/-); P = 0.0357, KC9A1 (WT) vs KC9A1 (NOS2-/-); P < 0.0001, STB-K (NOS2-/-) vs KC9A1 (NOS2-/-); P < 0.0001. Using this route of infection and the C57BL/6 mouse strain, the bacterial burden in the lungs and spleen at day 56 post infection was consistent with the enhanced persistence phenotype of KC9A1 when compared with STB-K. In NOS2-/- mice, the persistence of STB-K in the spleen and KC9A1 in both organs were increased showing that NO is an important factor reducing STB-K and KC9A1 persistence. However, the difference between STB-K and KC9A1 was maintained in NOS2-/- mice, and even increased in comparison to C57BL/6 mice, indicating that the rv1339 mutation also confers strong resistance to NOS2-/- independent mechanisms. c, BMDMs from BALB/c mice were either unstimulated, or stimulated with IFN-γ (100 U/ml) + LPS (100 ng/ml), or stimulated with IFN-γ + LPS and treated with the inducible nitric oxide synthase (iNOS) inhibitor L-NAME (4 mM). BMDMs (2.5 ×105 cells/well) were infected at a multiplicity of infection (MOI) of 0.5 bacteria/cell with STB-K, KC9A1, KC9C1 or MTB H37Rv and the number of surviving bacteria were evaluated by plating on 7H11 solid medium. The indicated value are the Log10 (ratio of bacterial load at day 3 over bacterial load at day 0 in the same experiment). *P < 0.05 by one-way repeated measure ANOVA with the Bonferroni post-hoc test. c) STB-K (IFN-γ + LPS) vs KC9A1 (IFN-γ + LPS); P = 0.0225, STB-K (IFN-γ + LPS) vs KC9C1 (IFN-γ + LPS); P = 0.0437. In these experiments, stimulation of BMDMs with IFN-γ and LPS reduced the survival of the 4 strains and this effect is partially suppressed by inhibiting iNOS.

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