Effect of pcaA on early colonization of the lung and its TNF dependence. (A) Wild-type C57BL/6 mice were infected by aerosol with wild-type Mtb (black bars) or the ΔpcaA mutant (open bars), and bacterial titers were determined at the indicated time points by serial dilution and plating of lung homogenates. Each bar is the mean of bacterial loads from four mice per group and error bars are SEM. When error bars are not visible they are within the plotted bar. P values (Student's t test) for the comparison of WT vs ΔpcaA mutant Mtb are indicated above each bar set. The 1- and 7-d time points at the far right are a repeat experiment including a complemented pcaA mutant (comp, hatched bar). (B) TNF^−/− mice were infected with either wild-type or ΔpcaA mutant Mtb as in A, and CFUs of lung tissue were determined at the indicated times. Each bar is the mean of four mice per group. (C) Survival kinetics for TNF^−/− mice infected with wild-type or ΔpcaA Mtb. 10 mice each were infected with either wild-type (black line, square symbol) or ΔpcaA Mtb (gray line, triangle symbol) via the aerosol route and the time to death was recorded.

Extractable lipids are sufficient to transfer the hypoinflammatory phenotype of the pcaA mutant. Murine bone marrow– derived macrophages were infected with wild-type Mtb (black bar) or ΔpcaA mutant Mtb (open bars) as in Fig. 2 A. Bacteria were either untreated (native), delipidated with petroleum ether (−lipid), delipidated and reconstituted with lipids from the same strain (−lipid + self), or delipidated and reconstituted with lipids from the opposite strain (−lipid + opposite). TNF levels after 24 h of infection are shown. *, P < 0.001.

Effects of pcaA modification of TDM on innate immune recognition by macrophages. (A) RAW 264.7 murine macrophage cell line or murine bone marrow–derived macrophages were stimulated with vehicle (stippled bar), or a monolayer of wild-type Mtb TDM (black bar), pcaA mutant TDM (open bar), or complemented mutant TDM (striped bar). TNF (ng/ml) in culture supernatants was measured 48 h after stimulation. *, P ≤ 0.001. ^#, P = 0.03. (B) RAW 264.7 cells were stimulated with indicated TDMs for 3 h, incubated for 3 h with Brefeldin A, and stained with monoclonal antibody to TNF. In the upper left plot, the shaded histogram is vehicle stimulated cells, whereas the open histogram is LPS-stimulated cells. Numbers above each plot are the percentage of TNF-positive and -negative cells using the gates established on unstimulated cells in the upper left panel. P < 0.01 for the comparison of WT versus ΔpcaA TDM by t test. (C) Dose response curve for TNF induction by wild-type TDM (triangle), ΔpcaA mutant TDM (square), or complemented TDM (circle) from RAW cells measured after 24 h of stimulation.

pcaA-dependent modification of extractable lipids mediates temporally restricted macrophage activation. (A) Requirement of pcaA for early proinflammatory activation of macrophages by Mtb infection. Murine bone marrow–derived macrophages were left uninfected (stippled bar), or infected with wild-type Mtb (black bars), ΔpcaA mutant Mtb (open bars), or the complemented ΔpcaA mutant (striped bars). At the indicated time points after infection, TNF (top), and IL-6 (bottom) levels were determined by ELISA of culture supernatants. The same experiments were performed with delipidated bacteria, pictured on the right side of A. *, P ≤ 0.01. (B) Temporal sequence of pcaA-dependent macrophage activation. Murine bone marrow–derived macrophages were infected as described in A and supernatants were removed and replaced with fresh media every 24 h. Supernatants from the indicated time periods were assayed for TNF by ELISA. Stippled bar is uninfected, black bar is wild-type Mtb, and white bar is ΔpcaA mutant infected. **, P < 0.001. (C) Effect of pcaA on intracellular replication of Mtb in macrophages. Bone marrow–derived macrophages from wild-type C57BL/6 mice (WT) or TNF-deficient mice (TNF^−/−) were infected with wild-type Mtb (black bars) or the ΔpcaA mutant (open bars) and intracellular bacterial titers were determined at the indicated time points. ^#, P = 0.02.

Purification and characterization of wild-type and ΔpcaA mutant TDM. (A) Chemical structure of TDM. (B) Thin layer chromatography of purified TDM from wild-type and ΔpcaA mutant M. tuberculosis developed with chloroform/methanol/water (90:10:1, vol/vol/vol). (C) Mycolate subclass analysis of TDM from wild-type and pcaA mutant strains. ¹⁴C-labeled TDM-derived mycolates were analyzed by high performance thin layer chromatography developed by running three times with hexane/ethyl acetate (95:5, vol/vol). Mycolate subclasses (α, methoxy, and ketomycolates) are labeled with arrowheads.

Reduced pulmonary granuloma formation by ΔpcaA TDM in mice. C57BL/6 mice were injected intravenously with a water–oil–water emulsion of TDM purified from wild-type (two left panels) or ΔpcaA mutant (two right panels) Mtb at a dose of 300 μg per mouse. The histology of the lungs was examined at 7, 14, and 21 d after injection. The area of granulomatous inflammation was quantitated as described in Materials and methods and plotted against time. Diamonds represent wild-type TDM whereas squares represent ΔpcaA mutant TDM. *, P < 0.05.