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. Author manuscript; available in PMC: 2019 Mar 17.
Published in final edited form as: Nat Microbiol. 2018 Sep 17;3(10):1099–1108. doi: 10.1038/s41564-018-0245-0

Figure 4. W-Beijing Mtb strains carrying the rpoB-H445Y SNP overexpress PDIM and bypass the IL-1R1 signaling pathway for protective immunity.

Figure 4.

B6 and Il1r1−/− macrophages were infected with HN878 or three independently isolated HN878 rpoB-S450L or HN878 rpoB-H445Y mutants (MOI1) for 6 days (n=4). IL-1β, lactate, and IFN-β levels were measured in supernatants (a) and intracellular CFU was determined (b). B6 and Il1r1−/− mice (n=5) were aerosol infected with 100 CFU Mtb HN878 rpoB-S450L or HN878 rpoB-H445Y. Lung bacterial burden was determined on 30 dpi (c). The LC/MS reconstructed ion chromatogram (RIC, m/z 1330–1450) of PDIM (d) and the internal standard TAG (e) from HN878 and W_7642 are overlayed (the two earlier elution peaks of the TAG chromatogram are from endogenous Mtb 18:1/16:0/26-TAG (second isotope) and 18:0/16:0/26:0-TAG). The amounts of 20:0/20:0/20:0-TAG internal standard in the two samples are nearly equal. The LC/MS RIC (m/z 1330–1450) of PDIM (f) and the internal standard TAG (g) from rpoB-H445Y and rpoB-S450L are overlayed. The 20:0/20:0/20:0-TAG internal standard in the rpoB-S450L sample is about 1.5 times of the amount in rpoB-H445Y. Trace data is representative of at least two replicates. PDIM was isolated from each Mtb strain and coated onto polystyrene beads. B6 macrophages (n=4, except rpoB-H445Y and rpoB-S450L PDIM n=8) were treated with PDIM coated beads (200:1) from different Mtb strains or control HSA coated beads (200:1) alone or in combination with HN878 infection (MOI1). IL-1β, lactate and IFN-β protein levels were determined in 6 dpi supernatants (h). UN-untreated, UI-uninfected, nd-not detectable. (a-c) 2-way ANOVA with Bonferroni post-test, (h) 1-way ANOVA with Tukey’s post-test. The data points represent the mean (±SD) of values. *p≤0.05, ***p≤0.001, ns-not significant (p>0.05).