Fig. 1.
Host-associated lipids support growth of M. tuberculosis in acidic media. (A) Growth of M. tuberculosis in glycerol and glucose containing media is restricted below pH 5.8. Biomass was measured (OD580nm) after 12 d of growth in media of decreasing pH containing 0.2% glucose and 0.2% glycerol. (B) Repeated OA supplementation supports continued growth of M. tuberculosis at pH 5.5. A total of 200 μM OA was used to replenish a FA-free 7H9 medium base containing 0.2% glycerol and 0.2% glucose. (C) pH measurements of the culture media (pHEX) during the growth curve experiment in B. (D–G) Growth at pH 5.5 is supported by repeated supplementation of (D) cholesterol (100 μM), (E) palmitic acid (100 μM), (F) OA (200 μM), a combination of (E) cholesterol + palmitic acid, (F) cholesterol + OA, and (G) arachidonic acid (100 μM). In B and D through G, arrows indicate when lipid(s) were added to cultures. (H) Growth of M. tuberculosis using repeated supplementation of 200 μM OA (or 50 μM OA for pH 4.5) across a range of decreasing pH. Base media was FA-free 7H9. (I) pH measurements of the culture media (pHEX) during the growth curve experiment described in H. Tyl: ETOH is added to the cultures (D–G) as a control vehicle used as a solvent for the lipids tested (gray, dashed). Data in B–I show means ± SD of three biological replicates. Statistical significance was determined for the final time point of growth curve experiments using one-way ANOVA and Tukey’s multiple comparison test. Data in A are means ± SD of two independent experiments performed in triplicate. In A, statistical significance was determined at each pH compared to pH 6.6. In B through G, black asterisks indicates significance relative to controls (FA-free, Tyl:EtOH); orange and pink asterisks indicate significance relative to data graphed in that same color. Gly = glycerol; OA = oleic acid; FA = fatty acid; Chol = cholesterol; PA = palmitic acid; Tyl: ETOH = tyloxapol-ethanol. ***P < 0.0005; ****P < 0.0001.