system, like infection with HIV, increases the chance of people becoming infected with TB. Mtb is persistent due to the fact that it infects the aveolar macrophages. While in the macrophage's phagosome in order to avoid its destruction, Mtb initiates formation of granulomas and it is capable of shutting down its metabolism and replication in order to transits to its dormant stage. While in the dormant stage Mtb is highly unaffected by drug treatment and is resistant to host immune system. (1, 2). The structure of the mycobacterial cell wall and envelope allows the bacillus to enter macrophages by employing multiple receptors (3). After the bacillus enters the macrophage it induces phagosome maturation which normally leads to the formation of phago-lysosome and decrease the pH to acidic pH~4.5. However Mtb prevents the phago-lysosome formation and keeps the pH levels normal and switches its metabolic pathways to utilize fatty acids thus leading to the survival of the bacillus in the macrophage.(12) The exact molecular mechanisms by which the mycobacteria prevents phago-lysosome formation are not yet fully understood, however it is known that cholesterol is accumulated at the site of entry and by depleting plasma membrane cholesterol it inhibits the uptake of Mtb. (4) Thus, by entering host cells at cholesterol-rich domains of the plasma membrane, mycobacteria may ensure their subsequent intracellular survival. Once in the phagosome the Mtb needs to sustain its nutrient uptake. (5, 6). Due to the fact that Mtb like some bacteria, does not synthesis sterols, it needs to employ cholesterol from the host in order to use it as a main source of carbon. Cholesterol has been shown to be required by Mtb during infection of macrophages. Therefore the host cholesterol biosynthesis pathway plays key role in the virulence and persistence of Mycobacterium tuberculosis. In this review will discuss the involvement of cholesterol in the virulence of Mtb and how the cholesterol metabolic pathway can be used for the development of novel therapeutics against tuberculosis.(7)
The Cholesterol pathway in Mycobacterium tuberculosis
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Cholesterol is an extremely important structural component in living cells. It has a major role in the formation of membrane structures, it is anabolic precursor for the biosynthesis of bile acids, vitamin D and steroid hormones and also plays a key role in the signal transduction pathways.(8). In animals cholesterol is mainly located in the membranes. The highest proportion of unesterified cholesterol is in the plasma membrane (roughly 30-50% of the lipid in the membrane or 60-80% of the cholesterol in the cell). Small quantities of cholesterol are also produced in plants (mainly as a precursor for some plant hormones). (9) However unlike animals, plants and fungi, most bacteria do not synthesis cholesterol. Nevertheless Mtb expresses essential genes which encode enzymes (e.g cytochrome P450) which plays important step in the cholesterol biosynthesis pathway. (10).
Mostly cholesterol in humans is biosynthesised de novo. The cytoplasm and the microsomes of the endoplasmic reticulum synthesis cholesterol from acetyl-CoA. Figure 1.
Figure 1: Cholesterol biosynthesis pathway
Metabolic pathways of Mtb important during infection. Growing evidence suggests that pathogenic mycobacteria rely on lipids in vivo.
Uptake of cholesterol by Mtb
Recently it was found that cholesterol uptake requires Mce4 transport system of M. tuberculosis and is regulated by the KstR. KstR is a TetR-type transcriptional receptor that controls the expression of genes involved in cholesterol utilization.(13, 14) The TetR-type transcription receptors co-ordinately regulate over 70 genes that are all de-repressed by growth on cholesterol.(13, 15) recent studies have shown that deletion of mce4 operon restricts mycobacteria growth in cholesterol rich environment.(16). Mce4 is also important during mycobacterium replication cycle while the macrophage is stimulated by IFN- γ.(17).
The first step in the M. tuberculosis cholesterol pathway is the oxidation of cholesterol into cholestenone by ChoD by reduction of NAD+ to NADH where ChoD is dehydrogenase protein.(18, 19, 20). The next step is the catabolism of cholesterol which is divided into two stages: the initial stage - degradation of alkyl side chain and subsequent - the cleavage of the steroid body. (Figure 2.) (21). In M. tuberculosis the accumulation of cholestenone is a result of obstruction of the side chain degradation whiche therefore suggests that the initial stage is the ring degradation stage which involves enzymes such as KsaAB and Has A-C.(22).
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Figure 2. Sterol side chain and ring chain degradation pathway of cholesterol in Mtb
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Side chain degradation
This side chain which is involved in the bile acid, aldehyde and alcohol production is reduced by hydroxylation of C26 and β-oxidation reactions which is mediated by cytochrome P450 (23). There are three major cytochrome P450 enzymes which can oxidize the side chain of cholesterol and cholestenone. Those are CYP124 encoded byRv2266, CYP125 encoded by Rv3545c, and CYP142 encoded by Rv3518c.The final ATP-dependent phase is catalyzed by a sterol-CoA ligase (7, 27). This is confirmed by recently identifying Mtb genes which encode β-oxidation enzymes. Such genes are the ltp2, fadE29, fadE28, fadA5, fadE30, FadE32, fadE33, fadE34, and hsd4B which encode thiolase enzyme.The thiolase enzyme catalyzes the thiolysis of acetoacetyl - CoA and is required for Mtb growth in cholesterol rich environment.(24). Over the years studies have shown that deletion of P450 and more specifically the CYP125 ans CYP124 leads to the inhibition of M. tuberculosis growth due to the fact that it cannot use cholesterol as its exclusive carbon source.(28)
Ring chain degradation
The initial phase during the sterol ring degradation is the conversion of cholesterol to cholestenon i.e. choles-4-en-3-one. This conversion is catalysed by 3β-hydroxysteroid dehydrogenase (3β-HSD) or by cholesterol oxidase (ChoD). (7). M. tuberculosis expresses a gene (Rv1106c) which encodes for the 3β-HSD enzyme. This enzyme is dehydrogenase enzyme and uses NAD+ to NADH reduction as a co-factor in order to qxidize cholesterol to the 3-keto-4-ene cholesterol product. (20). While Rv1106c encodes for 3β-HSD, Rv3409c encodes for cholesterol oxidase which is involved in the second pathway by which the sterol ring chain can be degraded.(18).
In Mtb, the hsaACDB genes are part of a single operon within the cholesterol regulon (15). The hsaA and hsaB genes encode an oxygenase and a reductase, respectively. HsaA and hsaB act as a flavin-dependent monooxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to the catechol 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA) (7, 25). Consequently deletion of hsaA and hsaB results in the disability of M. tuberculosis to grow in activated macrophages and in cholesterol environment. Another gene of Mtb that has been identified as required for growth in macrophages is hsaD. HsaD is a member of the α/β hydrolase family. It is involved in the aerobic degradation of aromatic compounds in mycobacteria. hsaD catalyzes hydrolytic bond cleavage of4,9-DSHA to 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and 2-hydroxy-hexa-2,4-dienoic acid (HHD) (7, 26). Then as a final step HHD is metabolized to tricarboxylic acid cycle intermediates and propionyl-CoA
Cholesterol toxicity and inhibition
Inhibition of the cholesterol metabolic pathways can cause carbon starvation but but also mycobacterium cell death. Due to the fact that Mycobacterium tuberculosis have need of cholesterol in order to grow and cause persistent infection, inhibition of some of its major transporters proteins can be an opportunity for development of novel therapeutics. Such a transporter that can be used for the development of new therapeutics is Mce4. Mce4 proteins may be involved in maintaining the Mtb in a nutrient deficient environment for long time due to the fact that it allows M. tuberculosis to use cholesterol as it energy and carbon source. The hypothesis that mce4 is involved in the mycobacterium cholesterol uptake is confirmed by recent studies which show that M.tuberculosis strains that lack mce4 operon show significantly reduced ability to use cholesterol as main carbon source.(16). Another good target could be KstR protein regulators. KstR regulate over 70 genes involved in the promotion of mycobacterium growth by using cholesterol as an energy source. Thereby inhibiting Kstr proteins those promoter genes would not be expressed therefore M.tuberculosis would not be able to persist inside the macrophages due to the fact that phago-lysosome formation would not be inhibited anymore. Thus making TB faster and easier to treat. Apart of Kstr and Mce4 a possible target to look at is cholestenone. There has been evidence that cholestenone as a primary product of the cholesterol biosynthesis pathway can be toxic and cause cell death.(22). Cholestenone is used by CYP125 which is P450 enzyme and has a major role in cholesterol side chain degradation. Therefore by knocking out P450 enzyme CYP125 the cholestenone accumulation in the cell increases and causes cell death. Moreover another study has discovered that deleting hsaC resulted in the formation of the harmful catechol.(29). The formation and accumulation of catechol causes the increase in free radicals inside the cell which cause irreparable DNA damage.(30). By inhibiting hsac and CYP125 will lead to cell poisoning itself to death which makes this approach probable for developing novel therapeutics against tuberculosis.
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Inhibiting CYP125 and HsaC may also be possible therapeutic options in which the cell poisons itself to death (16).
Cholesterol plays major role in cell signalling, cell trafficking, membrane structure and production of hormones. By the evidence stated above it can be concluded that Mycobacterium tuberculosis can store and utilize host cholesterol depending on the availability of the nutrients in the cell environment. (31). Furthermore the presence of cholesterol within the host macrophage plasma membrane promotes the formation of lipid rafts which enhance the bacilli entry.(4). After the entry the bacteria inhibits phagosome maturation by yet not completely understood mechanisms. However it has been postulated that removal of cholesterol from the phagosomes can prevent the bacterial phagosome inhibition thus allowing phagosome maturation which then leads to bacillus death.(32). From previous research it can be concluded that cholesterol plays a crucial role in mycobacteria binding to macrophages and in prevention of phago-lysosome fusion. Therefore tubercle bacilli becomes protected from host immune response and can cause latent or persistent infection.