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It is expected that the obligatory human pathogen Mycobacterium tuberculosis must adapt metabolically to the various nutrients available during its cycle of infection, persistence, and reactivation. Cholesterol, which is an important part of the mammalian cytoplasmic membrane, is a potential energy source. Here, we show that M. tuberculosis grown in medium containing a carbon source other than cholesterol is able to accumulate cholesterol in the free-lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary antituberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able to grow on mineral medium supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537 (kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of the intermediate, 9-hydroxy-4-androstene-3,17-dione. Our findings that M. tuberculosis is able to accumulate cholesterol in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future studies into the pathophysiology of this important deadly pathogen.
Mycobacterium tuberculosis, the causative agent of tuberculosis, is a very successful pathogen that infects one-third of the human population (21). Only 10% of primary infected individuals develop active disease during their lifetimes. Tubercle bacilli are able to persist in a dormant state, from which they may reactivate and induce the contagious disease state (13). In asymptomatic hosts, M. tuberculosis exists in reservoirs called granulomas, which are cellular aggregates that restrict bacterial spreading (40). Granulomas are organized collections of mature macrophages that exhibit a certain typical morphology and that arise in response to persistent intracellular pathogens (1, 4). Pathogenic mycobacteria can induce the formation of foamy macrophages filled with lipid-containing bodies; these have been postulated to act as a secure, nutrient-rich reservoir for tubercle bacilli (31). Moreover, M. tuberculosis DNA has been detected in fatty tissues surrounding the kidneys, as well as those of the stomach, lymph nodes, heart, and skin. Tubercle bacilli are able to enter adipocytes, where they accumulate within intracytoplasmic lipid inclusions and survive in a nonreplicating state (26). In vivo, it is expected that M. tuberculosis adapts metabolically to nutrient-poor conditions characterized by glucose deficiency and an abundance of fatty acids (25, 26). The presence of a complex repertoire of lipid metabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important alternative carbon and energy sources for this pathogen (7).
One attractive potential alternative nutrient that is readily available in the mammalian host is cholesterol, a major sterol of the plasma membrane. The presence of cholesterol in lipid rafts is required in order for microorganisms to enter the intracellular compartment (14). Studies have shown that cholesterol is essential for the uptake of mycobacteria by macrophages, and it has been found to accumulate at the site of M. tuberculosis entry (2, 12, 30). Moreover, cholesterol depletion overcomes the phagosome maturation block experienced by Mycobacterium avium-infected macrophages (10).
It is well known that cholesterol can be utilized by fast-growing, nonpathogenic mycobacteria (5, 20, 22), but it was previously thought that pathogenic mycobacteria might not be able to use cholesterol as a carbon and energy source (3). Recently, however, bioinformatic analysis identified a cassette of cholesterol catabolism genes in actinomycetes, including the M. tuberculosis complex (41). Microarray analysis of Rhodococcus sp. grown in the presence of cholesterol revealed the upregulation of 572 genes, most of which fell within six clearly discernible clusters (41). Most of the identified genes had significant homology to known steroid degradation genes from other organisms and were distributed within a single 51-gene cluster that appears to be very similar to a cluster present in the genome of M. tuberculosis (41). Many of the cholesterol-induced genes had been previously selected by transposon site hybridization analysis of genes that are essential for survival of tubercle bacilli (33) and/or are upregulated in gamma interferon-activated macrophages (37, 42). It was also demonstrated that the M. tuberculosis complex can grow on mineral medium with cholesterol as a primary source of carbon (27, 41). Moreover, the growth of tubercle bacilli on cholesterol was significantly affected by knockout of the mce4 gene, which encodes an ABC transporter responsible for cholesterol uptake (24, 27). Earlier studies had shown that disruption of mce4 attenuated bacterial growth in the spleens of infected animals that had developed adaptive immunity (17, 35).
In the present study, we demonstrate for the first time that M. tuberculosis utilizes cholesterol via the 4-androstene-3,17-dione/1,4-androstadiene-3,17-dione pathway (AD/ADD) and that this process requires production of an intact KstD enzyme. We also show that tubercle bacilli growing in medium containing an alternative carbon source can accumulate cholesterol in the free-lipid zone of their cell walls, and this accumulation affects cell wall permeability.
M. tuberculosis strain H37Rv was maintained on Middlebrook 7H10 agar or 7H9 broth supplemented with 10% OADC enrichment (oleic acid, albumin, dextrose, catalase) and 25 μg/ml kanamycin (Km) when required. For growth on defined carbon sources, strains were grown in minimal medium supplemented with 0.01% cholesterol, as described previously (27).
Cholesterol uptake by M. tuberculosis was monitored on 7H9-OADC medium in cultures of living and thermally killed mycobacterial cells. Tritiated 1α,2α(n)-[3H]cholesterol (activity of 35 to 50 Ci/mmol; Amersham Biosciences, United Kingdom) was added to the culture medium at a final concentration of 1 μCi/ml. During bacterial growth, 1-ml culture samples were taken at the indicated times (see Fig. Fig.1)1) (two samples per time point) and centrifuged at 16,000 × g for 15 min at 4°C. The cell pellets were washed twice and resuspended in Tris-EDTA buffer. Cells from the first sample were mixed with OptiPhase scintillation fluid (Perkin Elmer), and the mycobacterial cell-associated radioactivity was determined by liquid scintillation counting, using a 1450 Microbeta Plus Liquid Scintillation Counter (Perkin Elmer). The second cell pellet was disrupted using a Mini Beatbeater-8 (BioSpec Products), and the resulting sample was centrifuged at 16,000 × g for 15 min at 4°C. The emissions of the supernatant (cytosolic fraction) and pellet (cell wall fraction) were monitored as described above. The activity of each sample (as nCi) was calculated from the counts per minute, after correction for counter efficiency, using an online calculator (www4.gelifesciences.com).
In order to visualize cholesterol accumulation in the mycobacterial cells, we used the fluorescent dye filipin (Sigma-Aldrich). Filipin is a polyene macrolide antibiotic from Streptomyces filipinensis that binds specifically to cholesterol molecules; this binding causes a conformation change and emission of fluorescence. M. tuberculosis was cultured in 7H9-OADC medium with and without (background control) 0.01% cholesterol. After 72 h of incubation, samples from both cultures were centrifuged (16,000 × g for 15 min at 4°C), washed three times in Dulbecco's phosphate-buffered saline (PBS; pH 7.4), and fixed with 3% paraformaldehyde for 1 h at room temperature. The samples were then washed with PBS, and the paraformaldehyde was quenched by incubation with glycine (1.5 mg/ml in PBS) for 10 min at room temperature. The cells were stained with 0.05 mg/ml of filipin working solution in PBS for 45 min at room temperature in the dark, followed by three rinses with PBS and visualization by fluorescence microscopy using a Nikon Eclipse TE2000-U inverted microscope with a UV filter set (340- to 380-nm excitation; 430-nm long pass filter).
Tubercle bacilli were grown in 7H9-OADC medium supplemented with cholesterol (0.01%). To determine the accumulation of cholesterol in mycobacterial cells, 5-ml culture samples were withdrawn at 24-h time intervals. The bacterial cells were spun down, washed five times for removal of extracellular cholesterol, and extracted three times with an equal volume of chloroform. To quantify the accumulation of cholesterol, equal amounts of 4-androstene-3,11,17-trione (Sigma) were added to each sample as an internal standard, and samples were subjected to gas chromatography as previously described (5). To obtain samples of the M. tuberculosis cell wall free-lipid zone and defatted cells, pellets were obtained from 20 ml of M. tuberculosis culture, washed five times, and extracted three times with an equal volume of chloroform-methanol (2:1, vol/vol) for 48 h at room temperature on a rotatory shaker (200 rpm). The resulting mixture was centrifuged at 3,200 × g for 30 min at 4°C. The resulting pellet was composed by defatted cells. The extracts were combined and evaporated to dryness under nitrogen, and the obtained free lipids and defatted cells were separated on Merck silica gel 60 thin-layer chromatography plates using chloroform-methanol-water (65:25:4, vol/vol/vol) as a solvent. The positions of the separated compounds were detected by spraying the plates with a 10% ethanolic solution of molybdophosphoric acid, followed by heating for 10 min at 180°C. Postculture medium (20 ml) was filtered through a Synpor filter (pore diameter, 0.22 μm) and extracted three times with an equal volume of chloroform-methanol (2:1, vol/vol). The extracts were combined and evaporated to dryness under nitrogen, and lipids were analyzed by thin-layer chromatography, as previously described (19).
M. tuberculosis cells (wild type and ΔkstD mutant) were cultured in minimal medium supplemented with 0.01% cholesterol (27). To follow the process of cholesterol biotransformation and detect intermediates, 5-ml samples were withdrawn from the culture at 24-h intervals and extracted three times with an equal volume of chloroform. The extracts were dried under a vacuum, the residue was dissolved in 0.5 ml of acetone, and the isolated steroids were analyzed by gas chromatography as previously described (34).
Tritiated rifampin (4-methylpiperazine-3H; activity, 10 Ci/mmol; Moravek Biochemicals) was used to examine the cell wall permeability of M. tuberculosis cells, based on the protocol of Piddock et al. (32). Mycobacterial cells were grown to mid-logarithmic phase (A600 of 1 to 1.2) on 7H9-OADC medium with and without 200 mg/liter of cholesterol. Fifty milliliters of the culture was centrifuged at 6,010 × g for 15 min at 4°C. The cells were washed in 10 ml of 50 mM sodium phosphate buffer (pH 7), resuspended in the same buffer to an optical density at 600 nm of 8, and placed in a 37°C water bath for 10 min to equilibrate. The [3H]rifampin was added at a final concentration of 0.272 μg/ml (3.33 μCi/ml), and 500-μl samples were removed at various time intervals. The samples were mixed with 1 ml of 50 mM sodium phosphate buffer (pH 7) on ice and centrifuged at 16,000 × g for 15 min at 4°C. The resulting cell pellets were washed again in the same buffer, recentrifuged, and mixed with OptiPhase scintillation fluid (Perkin Elmer). The cell-associated radioactivity was determined by liquid scintillation counting, as described above. Passive adsorption of rifampin to the cell wall (background) was estimated by performing the experiments at 0°C; these results were subtracted from the values obtained at 37°C to determine the activity from rifampin that had actively accumulated in the cells.
Fifty-milliliter samples of M. tuberculosis culture (1 × 108 bacteria/ml) grown in 7H9 broth with or without cholesterol were spun down at 4,000 × g for 20 min at room temperature. Each mycobacterial pellet was washed once with PBS and then with PBS supplemented with 1% bovine serum albumin, and the cells were resuspended in the latter buffer to a density of 5 × 109 cells/ml. The cells were incubated with specific fluorescein isothiocyanate-labeled anti-M. tuberculosis antibodies (final dilution, 1:50; Abcam) for 1 h at 37°C with continuous mixing. Bacterial samples incubated without antibodies served as negative controls. The optimal working dilution of antimycobacterial antibodies was determined in preliminary titration experiments. After incubation, the experimental and control samples were washed with PBS-1% bovine serum albumin and resuspended in 100 μl of the same buffer, and fluorescence was determined using a Wallac Victor 2 reader. All samples were run in quadruplicate for two independent experiments.
To perform unmarked deletion of the kstD gene from M. tuberculosis, we used a suicidal recombination delivery vector based on p2NIL (28). The recombination vector carried the 5′ kstD upstream region (1,603 bp) and the first 20 bp of the kstD gene tagged to the 3′ part of the kstD gene (645 bp), followed by 1,009 bp of the kstD downstream region. PCR products carrying 5′ and 3′ fragments of the gene were ligated out of frame, such that the resulting ΔkstD gene encoded a nonfunctional protein. The final vector (pAB30) also included the screening cassette from pGOAL17 (28). A gene replacement strategy was used to disrupt kstD at its native locus on the chromosome. The plasmid DNA was treated with NaOH (0.2 mM) and integrated into the M. tuberculosis chromosome by homologous recombination. The resulting single-crossover homologous recombinant mutant colonies were blue, Kmr, and sensitive to sucrose. The site of recombination was confirmed by PCR and Southern blot hybridization. The single-crossover strains were further processed to select for double-crossover mutants, which were white, Kms, and resistant to sucrose (2%). PCR and Southern blot hybridization were used to distinguish between the wild-type and double-crossover mutants (see Fig. S1 in the supplemental material). To engineer the complementation construct, the kstD gene was amplified using M. tuberculosis genomic DNA as a template and cloned into the pMV261 shuttle vector (5) under the control of the Phsp promoter. Next, the intact gene and promoter were relocated into the pMV306 integration vector. The final construct, named pMVkstD, was electrotransformed and integrated into the attB site of the M. tuberculosis ΔkstD genome to complement the unmarked deletion of the kstD wild-type gene.
It is well known that fast-growing mycobacteria degrade natural sterols and use them as a source of carbon and energy (5, 20, 22). However, the ability of tubercle bacilli to utilize cholesterol was not observed until recently (27, 41). To determine conclusively whether M. tuberculosis could accumulate cholesterol, we followed the fate of tritium-labeled cholesterol supplemented into bacterial cultures. M. tuberculosis was grown in rich (7H9-OADC) medium, and radiolabeled cholesterol was added to living cells at early log phase and to thermally killed cells. Samples were withdrawn every 24 h and monitored for potential incorporation of cholesterol into tubercle bacilli. Bacterial cells were separated by centrifugation, washed carefully, and analyzed by scintillation counting. A significant time-dependent increase of radioactivity was observed in living cells but not in dead cells, indicating that M. tuberculosis actively incorporated cholesterol (Fig. (Fig.1a).1a). For identification of the preliminary destination of cholesterol in the bacilli, cells were mechanically disrupted, and cytosolic and cellular debris fractions were analyzed. The majority of the observed radioactivity was detected in the insoluble fraction containing cell wall fragments (Fig. (Fig.1b1b).
The cholesterol of the mammalian cell membrane can be visualized by the fluorescent dye filipin (11, 12, 27). Accordingly, we used this cholesterol-binding compound to label any cholesterol incorporated into mycobacteria. M. tuberculosis cells grown in the presence and absence of cholesterol were subjected to filipin staining, as described in the Materials and Methods section. Microscopic analysis revealed filipin binding of cells grown in the presence of cholesterol but not in control cells (Fig. (Fig.2).2). Analysis of individual bacilli indicated that the dye-bound cholesterol was deposited in the cell envelope, not intracellularly. Moreover, tubercle bacilli growing in the presence of cholesterol were treated with organic compounds to extract free lipids, the external layer of the cell wall. The defatted cells were stained in the presence of filipin and analyzed by microscopy. The dramatic decrease in fluorescence was observed by comparing defatted and control cells (Fig. (Fig.22).
As a more accurate way to identify cholesterol incorporation into bacteria, we next applied gas chromatography. Cells grown in the presence of cholesterol were collected at different time points and carefully washed, and steroids were organically extracted from these cells and analyzed in the presence of an internal standard. Our results revealed a time-dependent accumulation of cholesterol in tubercle bacilli, verifying the ability of M. tuberculosis grown in rich medium to accumulate cholesterol (Fig. (Fig.3A).3A). The above data showed that tubercle bacilli are able to store cholesterol, at least when grown in rich medium, and indicated that the cell wall is a potential site of cholesterol accumulation. We hypothesized that cholesterol could accumulate in the most external layer of the cell wall, the free-lipid zone, which is more loosely formed than the other parts of the mycobacterial cell wall. To verify this hypothesis, we isolated and carefully washed cells grown in the presence of cholesterol (or tritium-labeled cholesterol), and subjected them to extraction of the free-lipid zone. The obtained extracts and defatted cells were analyzed by thin-layer chromatography, which revealed that cholesterol was, indeed, deposited in the free-lipid zone, together with phospholipids, glycolipids, and sphingolipids (Fig. (Fig.3B).3B). The study of bacilli growing in the presence of tritium-labeled cholesterol revealed significant radioactivity in the free-lipid extracts but not in the defatted cells (see Fig. S2 in the supplemental material).
Having confirmed through multiple methods that M. tuberculosis cells can accumulate cholesterol in their cell walls, we next examined whether this ability has physiological consequences for the deadly pathogen. We hypothesized that accumulation of cholesterol might protect tubercle bacilli against toxic compounds by decreasing their cell wall permeability. To test this hypothesis, we used the first-line antituberculosis drug rifampin, which is administered to tuberculosis patients worldwide. A scintillation counter was utilized to monitor the uptake of tritiated rifampin by M. tuberculosis cells grown in the presence or absence of cholesterol. The obtained results clearly showed that accumulation of cholesterol by tubercle bacilli affected cell wall permeability, resulting in decreased uptake of rifampin (Fig. (Fig.44).
We also questioned whether the accumulation of cholesterol in the outer part of the cell wall could mask the surface antigens of tubercle bacilli. Using fluorescein isothiocyanate-labeled antibodies, we quantitatively examined whether the recognition of mycobacterial antigens differed between cells grown in the presence and absence of cholesterol and found that bacilli grown in the presence of cholesterol showed ~20% less specific antibody binding than an equal number of M. tuberculosis cells cultured without cholesterol (see Fig. S3 in the supplemental material).
A recent study using cholesterol radiolabeled with 14C at the 4 position of the A sterol ring showed that the labeled carbon was converted to CO2 when the cholesterol was added to M. tuberculosis culture and that this process was dependent on the Mce4 cholesterol transporter (27). We therefore wondered if utilization of cholesterol by M. tuberculosis could be carried out by the AD/ADD pathway and if 3-ketosteroid Δ1-dehydrogenase (KstD) is essential for this process. We reported previously that KstD is essential for cholesterol utilization by Mycobacterium smegmatis and that M. smegmatis ΔkstD can be efficiently complemented with the kstD of M. tuberculosis delivered via a plasmid (5). More recently, KstD of M. tuberculosis was expressed in Escherichia coli, purified, and analyzed biochemically (18). The two-step recombination protocol of Parish and Stoker (28) was used to delete the kstD gene from the M. tuberculosis chromosome, as described in the Materials and Methods section. The resulting mutant was verified by PCR and Southern blot hybridization (see Fig. S1 in the supplemental material). The growth of the engineered strain in rich medium was not significantly different from that of the wild-type strain. To determine whether KstD is essential for cholesterol utilization, we compared the ability of wild-type M. tuberculosis, the ΔkstD mutant, and the ΔkstD mutant complemented with an intact kstD gene controlled by the heat shock protein promoter (ΔkstD-PhspkstD) to grow using cholesterol as the sole source of carbon and energy. We found that M. tuberculosis, but not its ΔkstD mutant, was able to use cholesterol as a primary source of carbon (Fig. (Fig.5A).5A). As we reported previously, the unmarked deletion of kstD in M. smegmatis resulted in the accumulation of the cholesterol degradation intermediates, AD and 9-hydroxy-4-androstene-3,17-dione (9OHAD), which could not be further degraded by the mutant lacking intact KstD (5). Consistent with our findings in M. smegmatis, we observed that M. tuberculosis ΔkstD mutants grown on mineral medium supplemented with cholesterol as the sole carbon source accumulated 9OHAD in a time-dependent manner, whereas this intermediate was not observed in wild-type and ΔkstD-PhspkstD cultures grown in the same medium (Fig. (Fig.5B).5B). Moreover, the time-dependent accumulation of 9OHAD was affected by supplementation of glycerol as an alternative carbon source (Fig. (Fig.5C).5C). Both the decreased growth of the ΔkstD mutant and its accumulation of 9OHAD on mineral medium supplemented with cholesterol are consistent with a previous report (27) that M. tuberculosis can utilize cholesterol as a carbon and energy source. Moreover, we also found direct evidence that cholesterol degradation in M. tuberculosis is performed exclusively by AD/ADD intermediates, with KstD playing an essential role in this process.
Cholesterol is an important membrane component in mammalian cells, where it plays well-documented roles in structure, signaling, and trafficking (15, 16, 23, 38). We herein demonstrate that M. tuberculosis can both accumulate and utilize cholesterol, depending on nutrient availability. Moreover, we found that cholesterol accumulation can change the cell wall permeability of the bacillus, and cholesterol utilization requires an intact KstD enzyme. The abilities of M. tuberculosis identified here by in vitro study cannot be directly applied to a discussion of the pathogenic process; however, our findings join a growing body of evidence suggesting that cholesterol may play a role in the pathogenesis of tuberculosis. Soon after inhalation, tubercle bacilli are phagocytosed by alveolar macrophages. The uptake of mycobacteria depends on the presence of cholesterol within plasma membrane lipids rafts, which accumulate at the site of mycobacterial entry (12, 30). Pathogenic mycobacteria are able to survive in the phagosomes of their host macrophages, which do not fuse with lysosomes due to maturation inhibition (8, 39). It has been postulated that the blockade of phagosome maturation requires direct contact of the phagosome membrane with the entire mycobacterial surface (9). Depletion of cholesterol from phagosomes infected with M. avium has been shown to lift the inhibition of maturation and allow phago-lysosome fusion (9). Thus, cholesterol binding seems to be crucial during the phagocytosis and intracellular survival of M. tuberculosis. The accumulation of cholesterol at these steps of infection would give tubercle bacilli an advantage within the host. As we have presented herein, cholesterol accumulation changed the cell wall permeability of the bacillus, inhibiting the in vitro uptake of the toxic compound, rifampin (a major antituberculosis drug). Moreover, cholesterol accumulation at least partially masked surface antigens in vitro, suggesting that it could help shield mycobacteria from the host immune system.
Macrophages that are infected with M. tuberculosis and prove unable to kill the intracellular pathogen will mature and aggregate to form granulomas containing lymphocytes, extracellular matrix components, calcifications, and caseous necrosis, which confine and eradicate the majority of tubercle bacilli (1, 31, 36). However, some bacilli are able to survive within the granuloma, resulting in a latent infection that can last the lifetime of an infected individual and may later reactivate as active tuberculosis.
It is not yet known whether cholesterol as a carbon and energy source could support long-term persistence of tubercle bacilli. As recently identified, M. tuberculosis possesses all the genes required to catabolize cholesterol to CO2 via the tricarboxylic acid cycle (41). Moreover, many of these genes, including kstD, appear to be inducible by cholesterol (41) and essential for survival of the bacillus in the macrophage (33, 29) and in vivo in mice (35), as identified by genome-wide screening. Mutations in the mce4 (cholesterol transporter) and choD (cholesterol oxidase) genes were found to attenuate M. tuberculosis in macrophages and in an in vivo mouse infection model (6, 27). Very recently, the hsaC gene (the iron-dependent extradiol dioxygenase responsible for opening of a ring A in cholesterol degradation) was found to attenuate M. tuberculosis in immunocompromised SCID mice and guinea pigs (43). Collectively, these findings seem to indicate that cholesterol transportation and utilization may be crucial to the fate of mycobacteria during the infection process. Here, we have directly shown that cholesterol ring structure degradation in M. tuberculosis occurs via the AD/ADD pathway, and disruption of kstD inhibits this process, leading to accumulation of intermediates. The requirement of kstD and hsaC for cholesterol degradation and survival of the bacilli in macrophages (29, 33, 43) strongly supports the hypothesis that cholesterol degradation is essential for the survival of tubercle bacilli during infection.
Based on the present and previous findings, we hypothesize that during the early stages of infection, pathogenic mycobacteria bind and accumulate host cholesterol to buffer their internalization into macrophages and inhibit phagosome maturation. This accumulation might change cell signaling by affecting lipid rafts and may protect tubercle bacilli against toxins by decreasing their cell wall permeability. Thus, when other nutrients may be less available, cholesterol can become the source of carbon and energy, thereby allowing tubercle bacilli to survive long-term in the host.
The work was supported partially by grants from ICGEB (contract CRP/POL07-01) and the State Committee for Scientific Research (contract N302 035 31/3172).
We are grateful to T. Parish for providing the p2NIL/pGOAL17 recombination system.
Published ahead of print on 28 August 2009.
†Supplemental material for this article may be found at http://jb.asm.org/.