Mycobacterial persistence requires the utilization of host cholesterol

Abstract

A hallmark of tuberculosis is the ability of the causative agent, Mycobacterium tuberculosis, to persist for decades despite a vigorous host immune response. Previously, we identified a mycobacterial gene cluster, mce4, that was specifically required for bacterial survival during this prolonged infection. We now show that mce4 encodes a cholesterol import system that enables M. tuberculosis to derive both carbon and energy from this ubiquitous component of host membranes. Cholesterol import is not required for establishing infection in mice or for growth in resting macrophages. However, this function is essential for persistence in the lungs of chronically infected animals and for growth within the IFN-gamma-activated macrophages that predominate at this stage of infection. This finding indicates that a major effect of IFN-gamma stimulation may be to sequester potential pathogens in a compartment devoid of more commonly used nutrients. The unusual capacity to catabolize sterols allows M. tuberculosis to circumvent this defense and thereby sustain a persistent infection.

Fig. 1.

M. tuberculosis catabolizes cholesterol. (A) Structure of cholesterol. The locations of the ¹⁴C label in substrates used in this study are indicated. (B) The conversion of [4-¹⁴C] or [26-¹⁴C]cholesterol to ¹⁴CO₂ by M. tuberculosis was quantified. Data are presented as the percentage of input radioactivity recovered as ¹⁴CO₂ after 4 h of culture. “Cold” indicates the addition of a 100-fold excess of unlabeled cholesterol. Error bars indicate standard deviation. (C) Cholesterol-derived carbon is assimilated into mycobacterial lipids. Bacteria were grown with cholesterol labeled at the indicated position, and the amounts of radioactivity recovered in the apolar or polar lipid extracts were determined. (D) The apolar lipid extracts of metabolically labeled bacteria were resolved by TLC. [3-¹⁴C]Propionate (Prop.) was used to specifically label branched-chain lipids such as PDIM (arrowheads). Labeled PDIM was detected in the [26-¹⁴C]cholesterol-labeled cells, but not in the [4-¹⁴C]cholesterol-labeled sample. The migration of intact cholesterol is shown (Chol.). The origin (asterisk) and direction of migration are indicated. The identity of PDIM was confirmed by purifying the species marked by arrowheads and subjecting them to atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Major ions characteristic of PDIM (45) were detected at m/z = 914, 928, 942, 956, and 970.

Fig. 2.

Cholesterol utilization requires Mce4 and MceG. (A) The indicated strains were cultured in defined medium containing 0.1% glycerol, 0.01% cholesterol, or no added carbon source. The OD₆₀₀ of each culture over time is plotted on a log scale. H37Rv is the parental strain of all mutants. (B) M. tuberculosis strains were incubated with [4-¹⁴C]cholesterol for the indicated times, and the cell-associated radioactivity remaining after extensive washing was quantified. Curves were fit by linear least squares regression. (C) The indicated strains were incubated with [4-¹⁴C]cholesterol for 4 h, and the relative production of ¹⁴CO₂ is plotted.

Fig. 3.

Cholesterol utilization is required for mycobacterial persistence in chronically infected mice and replication of IFN-γ-activated macrophages. (A) The abilities of wild-type and Δmce4 mutant bacteria to persist in mouse lungs were compared. C57BL/6 mice were infected i.v. with a 1:1 mixture of wild-type (H37Rv) and Δmce4 mutant bacteria, and the abundance of each strain in lung homogenates was quantified at the indicated times by plating. (B) The abilities of wild-type and mce mutants to replicate in bone marrow-derived macrophages (BMM) were compared. The number of cfu recovered from cell lysates at 0 and 5 days after infection are presented. The indicated macrophages were stimulated with IFN-γ 24 before infection. The data in A and B are representative of three or four independent experiments, respectively. Error bars indicate standard deviation. (C–F) Colocalization of cholesterol and M. tuberculosis in IFN-γ-activated macrophages. Filipin-stained cholesterol (C) and RFP-expressing bacteria (D) were simultaneously visualized in infected BMMs by using confocal microscopy. These images were merged to demonstrate colocalization (E, orange) and overlaid on a DIC image (F). In representative optical sections, 53–85% of RFP-positive pixels colocalized with filipin. The three fluorescent images shown represent a single optical section.