Indian Journal of Experimental Biology Vol. 47, June 2009, pp. 407-411 Review Article Role of cholesterol in Mycobacterium tuberculosis infection Maurine D Miner1, Jennifer C Chang1,2, Amit K Pandey3, Christopher M Sassetti3 & David R Sherman*1,4 1 Seattle Biomedical Research Institute, Seattle, WA 98109, USA Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA 3 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA 4 Interdisciplinary Program in Pathobiology, Department of Global Health, University of Washington, Seattle, WA 98195, USA 2 Mycobacterium tuberculosis (MTB) acquisition and utilization of nutrients within the host cell is poorly understood, although it has been hypothesized that host lipids probably play an important role in MTB survival. Cholesterol has recently been identified as an important lipid for mycobacterial infection. The mce4 transport system is required for cholesterol import into bacterial cells, and deletion of mce4 locus resulted in severe attenuation in a chronic mouse model of infection. However, it has remained unclear what additional bacterial functions were required for utilization of this sterol. We have found that the igr locus, which was previously found essential for intracellular growth and virulence of MTB, is required for cholesterol metabolism: igr-deficient bacteria cannot grow using cholesterol as a primary carbon source. The growthinhibitory effect of cholesterol in vitro depends on cholesterol import, as the Δigr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout the course of infection, and that degradation of this sterol is crucial for bacterial persistence. Keywords: Cholesterol, Δigr, Lipid metabolism, mce4, Mycobacterium tuberculosis Mycobacterium tuberculosis (MTB) infection is one of the leading causes of death worldwide. It kills an estimated 2 million people annually, and approximately 2 billion (one third of the world’s population) are infected with this organism. MTB resides in a modified phagosome of host macrophages and, like other intracellular pathogens, has adapted unique ways of thriving in this harsh environment. While several genes and virulence factors have been studied extensively, very little is known about the ability of MTB to infect a host and cause latent infections for years and sometimes decade1-5. The most fundamental challenge MTB faces in the host is the need to acquire nutrients from host cells. It is not clear what nutrients MTB obtains from the host in order to survive in the harsh surroundings of the nutrient-deficient vacuole, although host lipids have been implicated as an important source of carbon. This was first suggested by the observation that fatty acids but not carbohydrates stimulate respiration of MTB isolated from mouse lung6. Subsequently, sequencing of the MTB genome revealed at least 250 genes potentially involved in lipid metabolism7. _____________ *Correspondent author E-mail: david.sherman@sbri.org Many of these genes are transcriptionally induced during intracellular growth, and a few are known to be required for infection8-12. However, the complexity of MTB lipid metabolism has made it difficult to determine if any individual gene is genuinely required for host lipid catabolism, as opposed to the synthesis or modification of an endogenous bacterial lipid. The array of lipids an MTB bacterium might encounter during the course of infection is currently unknown. MTB likely interacts with fatty acids in the macrophage vacuolar membrane. Phagosomal membranes are complex structures in themselves, the bacteria most likely acquire nutrients from cytosolic molecules as well. The role of these fatty acids affecting MTB growth, and the ability of the pathogen to cause infection and disease, is currently of great interest. Major questions are a) which lipids are MTB exposed to during infection and b) how are those lipids utilized by the mycobacteria? The link between cholesterol and tuberculosis disease has been observed by an array of methods. Epidemiologic analysis has revealed a link between patient cholesterol levels and the outcome of pulmonary tuberculosis. These studies have concluded that higher serum cholesterol levels 408 INDIAN J EXP BIOL, JUNE 2009 correlated with reduced radiologic signs of disease and faster sputum sterilization following the initiation of chemotherapy13,14. Using molecular genetics and microbiology, the MTB-cholesterol connection has been further elucidated by Pieters et al.15, showing that cholesterol is required for at least one route of mycobacterial entry into macrophages. It was further shown that cholesterol is required for retention of the host protein coronin 1 (also called P57 or TACO) on mycobacteria-containing phagosomes and that retention of coronin 1 is associated with a block in phagosome-lysosome fusion16. Altogether these studies illustrate the importance of host cholesterol for MTB in the context of host macrophages and lung infection. However, the bacterial utilization of cholesterol during infection remained unresolved. Cholesterol import: the mce4 transport system— The mce4 locus is one of four homologous regions in the MTB genome and consists of several genes predicted to encode a multi-subunit ABC-like transport system17 (Fig. 1). Pandey and Sassetti recently identified cholesterol as a substrate for the mce4 transporter18. Upon deletion of the mce4 operon, MTB showed a marked growth defect in media containing cholesterol as the sole carbon source, as well as severely reduced accumulation of cholesterol compared to wild type. Cholesterol degradation by MTB can be monitored from both the 4- and 26-carbons of the molecule, with C-4 converted to CO2 and C-26 becoming incorporated into cell membrane lipids18. The Δmce4 strain was unable to convert radiolabeled cholesterol to CO2 or incorporate radiolabeled cholesterol into the cell membrane, consistent with the hypothesis that mce4 is required Fig. 1—The mce4 locus of M. tuberculosis. [Gene annotation and predictive function are based on Tuberculist—yellow box – cell wall processes; green box – virulence, detox and adaptation; orange box – conserved hypothetical] for MTB cholesterol import. This block in import resulted in the attenuation of Δmce4 bacteria in activated but not naïve macrophages, and severe attenuation in the mouse model of infection during late but not early time-points18. These studies show that cholesterol import is necessary for MTB during chronic infection, a time when host macrophages become INF-γ activated, possibly limiting nutrient availability. In a separate study of mce4, Joshi et al. used TraSH (Transposon Site Hybridization) to identify genes that genetically interact with the mce4 locus. Among the genes genetically linked to this transport system was the operon Rv3540-5c17. Bioinformatics suggested that these genes were involved in lipid metabolism9. The igr operon—After the TraSH analysis report17, another study showed that the Rv3540-5c operon was required for MTB growth in THP-1 cells in a tissue culture model of infection9. Due to the inability of transposon mutants with disruptions in Rv3540-5c to grow in macrophages, the operon was named igr for intracellular growth. Expression of this operon was previously found to be up-regulated in macrophages where it plays an important role, and these genes were found to be required for survival in mouse spleens following intravenous infection10,19,20. The six genes of the igr operon are annotated to suggest some role in lipid metabolism: a putative cytochrome p450 (Rv3545c, igrA), two acyl-coA dehydrogenases (Rv3543-4c, igrBC), two conserved hypothetical proteins (Rv3541-2c, igrDE), and a lipid carrier protein (Rv3540c, igrF) (Fig. 2). It has been hypothesized that the igr deletion mutant could be defective in some aspect of lipid metabolism and therefore might grow atypically in the Fig. 2—The igr locus of M. tuberculosis. [Gene annotation and predictive function are based on Tuberculist—blue box – lipid metabolism (igrB: fadE28, igrC: fadE29, igrF: ltp2); beige box – intermediary metabolism (igrA: cyp125); and orange box – conserved hypothetical (igrD: Rv3542c, igrE: Rv3541c)] MINER et al.: M. TUBERCULOSIS & CHOLESTEROL presence of one or more lipids. The igr deletion mutant grew identically to wild type strains in media containing a variety of fatty acids, including pyruvate, succinate, butyrate, valerate, isovalerate, dodecanoate, palimitate, and Tween 80 (Ref 9). In addition, the igr deletion mutant showed no discernable difference from wild type in the presence of iron chelators, acidified nitrite or hydrogen peroxide, mechanisms used by macrophages to control MTB infection (Chang & Sherman, unpublished observations). Despite the similarities of Δigr and wild type in all these conditions, the Δigr strain was powerfully attenuated in vivo. Aerosol infection of mice revealed a severe lag in growth in the lungs and delayed dissemination to the spleen9 (Fig. 3). While colony forming units (CFUs) eventually reached wild type levels, the histopathology of lungs from Δigr infected mice showed considerably less damage than those infected with wild type9. The unusual delayed growth phenotype in mice showed that the igr locus is Fig. 3—Δigr attenuation in immunocompetent mice.—Female C57BL/6 mice were infected via aerosol with approximately 200 CFU of H37Rv (WT) (□), Δigr (◊), or complement (Δ). At the times indicated, bacterial burdens in (A) lungs or (B) spleens were determined by plating homogenized tissues. [The data shown represent the average mean SD for 4–5 mice/strain/time point. Adapted from Chang et al.9] 409 important during infection, but provided few clues as to the role that these genes play. igr and cholesterol—While the lipid substrate for the igr system remained a mystery, a breakthrough came from a publication describing a cholesterolcatabolism gene cluster in the MTB-related actinomycete Rhodococcus strain RHA121. The authors identified a 51-gene cluster of RHA1 whose transcription was specifically induced in the presence of cholesterol. This region corresponds to an 82-gene cluster in the genome of MTB that includes both the igr operon as well as the mce4 transport system21. In addition, conditions were identified in which MTB could grow, albeit slowly, with cholesterol as the sole carbon source. Considering the genetic link between igr genes and the mce4 cholesterol transport system, these data seemed to suggest the igr operon might be involved in cholesterol metabolism during MTB intracellular growth. Using the growth conditions described by Van der Geize et al.21 we compared growth of wild type and igr deletion strains in the presence of cholesterol. In preliminary experiments, the phenotype was striking: the igr deletion mutant was unable to grow in the presence of cholesterol. In addition, the growth arrest could not be rescued by the addition of a preferred carbon source, such as dextrose or glycerol (Chang et al., submitted). These data suggest that cholesterol metabolism is incomplete in the igr mutant bacilli and that cholesterol or one of its metabolites is toxic to these cells. To determine if a block in cholesterol import could rescue the growth arrest of Δigr in cholesterolcontaining media, an MTB mutant disrupted in both the igr and mce4 loci was constructed. In preliminary experiments, this double knock-out (KO) strain displayed a partial rescue of the igr-mutant phenotype, with improved growth kinetics in the presence of cholesterol or cholesterol + glycerol. In addition, the double mutant was able to grow in and kill THP-1 cells close to levels of wild type bacteria. Complementation was also evident in vivo. In mice infected with the double KO, deletion of the mce4 cholesterol transport system restored bacterial growth in the lungs and spleens to wild type levels out to 3 weeks post-infection (Chang et al., submitted). These data are consistent with a model in which attenuation of Δigr results from the inability to degrade cholesterol properly along with the subsequent buildup of toxic intermediates (Fig. 4). 410 INDIAN J EXP BIOL, JUNE 2009 Fig. 4—Role of mce4 and igr in cholesterol metabolism. —Model depicting the possible roles of the mce4 and igr operons in cholesterol uptake and metabolism. MTB infects host macrophages and resides in a modified vacuole (inset). Cholesterol is transported into MTB via the Mce4 transport system. C-4 carbon is metabolized to CO2 and C-26 carbon is incorporated into bacterial cell lipids. At some point during cholesterol metabolism, igr genes are necessary for further degradation. Lack of igr-dependent metabolism could result in the build-up of a toxic metabolite. Multiple arrows represent multiple steps. Final products of cholesterol metabolism in MTB are unknown (shown as question mark). Analysis of the Δigr and Δmce4 strains is helping to elucidate new roles for cholesterol during MTB infection. The Δigr strain shows marked attenuation at early time-points following infection of mice, and subsequently bacterial CFUs approch wild type level. In contrast, strains deficient in the ability to import cholesterol (Δmce4) are only attenuated in mice at late time-points, indicating that cholesterol metabolism becomes limiting for MTB growth/survival only during chronic infection, after adaptive immunity has altered the environment in which MTB resides. Taken together, these data suggest that cholesterol is available to MTB throughout the course of infection, but becomes a key nutrient during chronic infection when the host environment has been altered by the adaptive immune response. Of course, the precise manner in which cholesterol is utilized by MTB in vivo remains unclear. One possibility is that cholesterol may become a major carbon source for ATP production and macromolecular biosynthesis. Alternatively, cholesterol metabolism by MTB may contribute to production of a specific virulence factor and/or a disruption of normal host cell signalling. Exploring these possibilities will be an area of active investigation in future. 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