**3.2. Lipid body formation in** *M. tuberculosis* **is critically dependent on lipid droplets**

Host lipids from lipid droplets are used by the pathogen as substantial nutrient source. Middlebrook already demonstrated in the late 1940s that mycobacterial growth *in vitro* was enhanced by supplementation with oleic acid [54]. Host lipids play an important role during infection. They appear to be the primary carbon source for *M. tuberculosis* in vivo. Over the last years several groups have reported that *M. tuberculosis* within foamy macrophages produces lipid bodies, suggesting that they are able to accumulate host cell lipids [19,55]. Neyrolles et al. showed that mycobacterial growth inside adipocytes occurs only after the formation of lipid droplets in the host cell. This result emphasizes that *M. tuberculosis* is dependent upon TAG provided by lipid droplets in host cells [55]. In 2011 Daniel et al. finally demonstrated that *M. tuberculosis* inside foamy macrophages imports fatty acids derived from host TAG and incorporates them intact into bacterial TAG. Moreover the group proved the accumulation of TAG in lipid bodies [46].

The utilization of host lipids in vivo does not only promote survival but may also increases virulence and modulate the immune response to infection. Growth of *M. tuberculosis* on fatty acids such as such propionate or valerate during infection leads to increased production of the surface-exposed lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1 (SL-1) [56].

Cholesterol utilization was also identified to be required for mycobacterial persistence [57]. In 2008 Pandey and Sassetti found that *M. tuberculosis* can grow using cholesterol as a primary carbon source and that the mce4 transporter is required for cholesterol uptake. *M. tuberculo‐ sis* contains four homologous mce operons, mce1–mce4, which are thought to encode lipid transporters [57,58].

#### **3.3. Biosynthesis of TAG and formation of lipid bodies in** *M. tuberculosis*

Biosynthesis of TAG consists of the sequential esterification of the glycerol moiety with fatty acyl-residues by various acyltransferases. Fatty acid biosynthesis consists of the stepwise addition of acetyl groups, which are provided by acetyl-CoA. The initial step is the transfer of an acetyl group from acetyl-CoA to a small protein, called acyl carrier protein (ACP). In the following two-carbon fragments are added sequentially to yield fatty acids of the desired length. *M. tuberculosis* uses both type I and type II FAS systems for fatty acid elongation. The multifunctional FAS I enzyme (*Rv2524c*) catalyzes the de novo synthesis of C16- and C18-S-ACP. These fatty acids are converted to the CoA derivative and used primarily for the synthesis of membrane phospholipids. By continuous elongation of these fatty acids FAS I produces specifically the C20- and C26-S-ACP products, and these fatty acids are released as the CoA derivatives. The C20 fatty acid is transferred to the FAS II system for the synthesis of the verylong-chain mero segment of α-, methoxy-, and ketomycolic acids [64]. The transfer from the FAS I to the FAS II system occurs by a key condensing enzyme, the ketoacyl ACP synthase III (FabH). FabH catalyzes the decarboxylative condensation of malonyl-ACP with the acyl-CoA products of the FAS I system (Figure 2). Two distinct cyclopropane synthases, MmaA2 and PcaA introduce cyclopropane rings into the the growing acyl chain [64-66].

operon for cholesterol uptake (mce1). All *M. leprae* five *mce* genes were overexpressed during intracellular growth in mouse and human biopsies [59,60]. This observation suggests, that the intracellular bacilli population induces cholersterol uptake of the infected cell and subse‐

Cholesterol is also essential for uptake of *M. tuberculosis* and *M. leprae* in macrophages. Cholesterol accumulates at the site of mycobacterial entry in macrophages and promotes mycobacterial uptake. Cholesterol mediates the recruitment of TACO from the plasma membrane to the phagosome [61]. TACO, also termed as CORO1A, is a coat protein that prevents phagosome-lysosome fusion and thus degradation of mycobacteria in phagolyso‐ somes (Figure 4) [61,62]. This mechanism for the formation of TACO-coated phagosomes

**3.2. Lipid body formation in** *M. tuberculosis* **is critically dependent on lipid droplets**

Host lipids from lipid droplets are used by the pathogen as substantial nutrient source. Middlebrook already demonstrated in the late 1940s that mycobacterial growth *in vitro* was enhanced by supplementation with oleic acid [54]. Host lipids play an important role during infection. They appear to be the primary carbon source for *M. tuberculosis* in vivo. Over the last years several groups have reported that *M. tuberculosis* within foamy macrophages produces lipid bodies, suggesting that they are able to accumulate host cell lipids [19,55]. Neyrolles et al. showed that mycobacterial growth inside adipocytes occurs only after the formation of lipid droplets in the host cell. This result emphasizes that *M. tuberculosis* is dependent upon TAG provided by lipid droplets in host cells [55]. In 2011 Daniel et al. finally demonstrated that *M. tuberculosis* inside foamy macrophages imports fatty acids derived from host TAG and incorporates them intact into bacterial TAG. Moreover the group proved the accumulation of

The utilization of host lipids in vivo does not only promote survival but may also increases virulence and modulate the immune response to infection. Growth of *M. tuberculosis* on fatty acids such as such propionate or valerate during infection leads to increased production of the surface-exposed lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1

Cholesterol utilization was also identified to be required for mycobacterial persistence [57]. In 2008 Pandey and Sassetti found that *M. tuberculosis* can grow using cholesterol as a primary carbon source and that the mce4 transporter is required for cholesterol uptake. *M. tuberculo‐ sis* contains four homologous mce operons, mce1–mce4, which are thought to encode lipid

Biosynthesis of TAG consists of the sequential esterification of the glycerol moiety with fatty acyl-residues by various acyltransferases. Fatty acid biosynthesis consists of the stepwise addition of acetyl groups, which are provided by acetyl-CoA. The initial step is the transfer of an acetyl group from acetyl-CoA to a small protein, called acyl carrier protein (ACP). In the

**3.3. Biosynthesis of TAG and formation of lipid bodies in** *M. tuberculosis*

quently uses the stored cholesterol as carbon and energy source.

promotes intracellular survival [62,63].

36 Tuberculosis - Current Issues in Diagnosis and Management

TAG in lipid bodies [46].

(SL-1) [56].

transporters [57,58].

**Figure 2.** Fatty acid biosynthesis in *Mycobacterium tuberculosis*. The FAS-II elongation module uses the substrates R-CO-S-ACP and malonyl-S-ACP derived from malonyl-S-CoA, generated by FabD. FabH condenses both substrates R, long-chain alkyl group. Enzymes involved in these reactions are as follows: FabG1, a β-ketoacyl-ACP reductase catalyz‐ es the reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP. β-hydroxyacyl-ACP dehydrase. 2-trans-eno‐ yl-ACP reductase (InhA). The β-ketoacyl-ACP synthase (KasA/KasB) catalyzes the addition of of two carbons from malonyl-ACP to R-CO-S-ACP (See text for details). R, long-chain alkyl group. ACP, acyl carrier protein. Enzymes are in bold letters. Selected inhibitors are depicted in red bold letters. TLM, thiolactomycin. CER, cerulenin. ETH, ethionamide. INH, isoniazid. TRC, triclosan. TAC, thiacetazone.

Esterification of fatty acids with glycerol-3-phosphate occurs via sequential acylation of the sn-1,2 and 3 positions of glycerol-3-phosphate, and removal of the phosphate group before the last acylation step. The terminal reaction is the esterification of diacylglycerol (DAG) with acyl-CoA by an diacylglycerol acyltransferase [40]. Animals and plants use diacylglycerol acyl‐transferases (DGAT) for the terminal esterification. DGATs catalyze ex‐ clusively the esterification of acyl-CoA with diacylglycerol. Bacteria do not contain DGATs but only bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferases (WS/DGAT). WS/DGATs, mediate next to TAG formation the synthesis of waxes by ester‐ ification of acyl-CoA with alcohol [67]. The genome of *M. tuberculosis* codes for 15 genes which contain the highly conserved putative active site motif of WS/DGATs (HHxxxDG). These genes were designated as "tgs", triacylglycerol synthases, but have only a weak se‐ quence similarity to other WS/DGAT sequences. All 15 expressed mycobacterial Tgs pro‐ teins show diacylglycerol acyltransferase activity and Tgs1 has the highest activity of all enzymes [48]. Gene disruption of *tgs1* results in a drastic reduction of major C26 longchain fatty acid in *M. tuberculosis* grown under hypoxic conditions. Thus Tgs1 appears to be a major contributor to TAG synthesis in *M. tuberculosis* so far [48,68]. And moreover two homologous proteins to Tgs1 and Tgs2 (BCG3153c and BCG3794c) and another poor‐ ly characterized acyltransferase (BCG1489c) were found to be exclusively associated to lip‐ id bodies. The disruption of *BCG3153c, BCG3794c*, and *BCG1489c* reduces TAG accumulation during the hypoxia-induced nonreplicating state, revealing that the en‐ zymes are involved in TAG synthesis during latency and pathogenicity [69].

The *M. leprae* genome shows only one predicted gene product which has has a significant degree of identity to any the Tgs enzymes from *M. tuberculosis* [18]. The tgs gene product ML1244 shows 72% identity to Rv2484c from *M. tuberculosis*. *Rv2448* is located next to a carboxylesterase lipQ, (*Rv2485c*), a probable glycerol-3-phosphate acyltransferase, (*Rv2482c*), a lysophosphatidic acid acyltransferase-like protein (*Rv2483c*), and a probable enoyl-CoA hydratase (*Rv2486*). The gene cluster of lipid metabolism genes suggests a possible involve‐ ment of the gene products in the synthesis of TAG [18]. A few tgs genes (*Rv3234c*, *Rv3233c*, *Rv2285*, and *Rv1425*) are located proximal to lipoproteins, which may serve as donors or

Lipid Inclusions in Mycobacterial Infections http://dx.doi.org/10.5772/54526 39

Neutral lipids in the core of the lipid body are hydrolyzed by lipases or esterases, yielding

In the genome of *M. tuberculosis* H37Rv twenty-one genes are termed as putative lipases (*lip* A to W, except K and S) [72]. The annotation was only based on the presence of the consensus sequence GXSXG, which is characteristic for the large group of the α/β hydro‐ lase fold protein family, which includes lipases as well as esterases, proteases, peroxidas‐ es, epoxide hydrolases and dehalogenases [72]. Thus the members of the lip group have only a very low level of sequence identity of ~20% and might have another function apart from lipid hydrolysis. Only the gene product of *Rv3097c* (LipY)) shows reasonable hydro‐ lase activity for long-chain TAG with chain lengths ranging from C4 to C18. Overexpres‐ sion of LipY induces extensive TAG hydrolysis. Disruption of *lipY* markedly reduces but does not completely inactivate TAG hydrolase activity, which suggests the presence of

Overexpression of *LipY* in *M. bovis* Bacillus Calmette-Guérin reduces protection against infection in mice, indicating that lipY plays a central role in TAG hydrolysis and virulence [47,73,74]. LipY contains a PE (Pro-Glu) domain, that is involved in modulation of LipY activity [73]. The PE domain contains a signal sequence for secretion of LipY by the ESX-5 system. It has been implicated that the secreted LipY is loosely associated with the bacterial surface where

Several other esterases, next to the members of the Lip group have been identified and biochemically characterized. They all belong also to the α/β hydrolase fold family and showing the minimal GXSXG motif. In 2007 Côtes et al. characterized a novel lipase Rv0183. The enzyme is only found in the cell wall and culture medium. This observation suggests that Rv0183 is involved in the degradation of the host cell lipids e.g. when M. tuberculosis infects adipocytes [55,76]. Another probably cell wall-associated carboxylesterase is encoded by Rv2224c. The esterase Rv2224c was found to be required for bacterial survival in mice [77]. The substrate spectrum of Rv2224c is poorly characterized and until now it is unknown whether the enzyme uses TAG as substrate [77]. Furthermore the three-dimensional structures of the esterases Rv0045c (PDB 3P2M) [78], Rv1847 (PDB 3S4K), and LipW (3QH4) from *M. tuberculosis* have been determined, but unfortunately it is not known whether these enzymes are involved in

**3.4. Activation of TAG – Lipases and esterases of** *M. tuberculosis*

fatty acids for energy generation and anabolism of membrane phospholipids.

acceptors of fatty acids [48]

other lipases in *M. tuberculosis* [47,49].

it may hydrolyze host's TAG [75].

TAG hydrolysis.

Ten of the 15 tgs genes in *M. tuberculosis* are located adjacent or proximal to 11 lip genes that are annotated as probable phospholipases or lipases-esterases-carboxylesterases. Some tgs genes may be cotranscribed with neighboring lip genes and may synthesize triacylglycerols from the released fatty acids from the host [18]. Lip gene products may be important for utilization of TAGs during dormancy and upon reactivation after dormancy. The tgs gene Rv0221 is located near lipC (*Rv0220*), lipW (*Rv0217c*), acyl-CoA synthetase (*Rv0214*), acyl-CoA dehydrogenase (*Rv0215c*), and an integral membrane acyltransferase (*Rv0228*). This clustering of genes of the fatty acid metabolism suggests that these genes may be cotranscribed and may release fatty acid from host TAG, carry out the transport of fatty acids and finally catalyze the re-synthesis of TAGs in the pathogen. Rv0221 and LipC have to be shown to be catalytical active. [18,70].

In summary Tgs enzymes play a major role in TAG synthesis, lipid body formation and maintenance.

Ag85A, a mycoltransferase, that is known to catalyze the formation of the cord factor was recently found to have additional DGAT activity [71]. The kinetic parameters are quite similar to those reported for the *M. tuberculosis* Tgs1-4, but the primary sequence of Ag85A does not contain the active site motif of WS/DGATs or TGS enzymes (HHxxxDG) [48,68,71]. Ag85A belongs to the α/β hydrolase fold family and contains the consensus GXSXG sequence. The enzyme is a carboxylesterase with an additional acyltransferase activity. Overexpression of Ag85A induces lipid body formation in *M. smegmatis*. The enzyme is located in the mycobac‐ terial cell wall, suggesting that it may be involved in the maintenance of lipid droplets in the host cell [71].

The genome of *M.leprae* contains also mycolytransferase 85 complex genes (A, B and C). Transcripts of these genes are upregulated either in infected nude mouse or human skin lesions [59].

The *M. leprae* genome shows only one predicted gene product which has has a significant degree of identity to any the Tgs enzymes from *M. tuberculosis* [18]. The tgs gene product ML1244 shows 72% identity to Rv2484c from *M. tuberculosis*. *Rv2448* is located next to a carboxylesterase lipQ, (*Rv2485c*), a probable glycerol-3-phosphate acyltransferase, (*Rv2482c*), a lysophosphatidic acid acyltransferase-like protein (*Rv2483c*), and a probable enoyl-CoA hydratase (*Rv2486*). The gene cluster of lipid metabolism genes suggests a possible involve‐ ment of the gene products in the synthesis of TAG [18]. A few tgs genes (*Rv3234c*, *Rv3233c*, *Rv2285*, and *Rv1425*) are located proximal to lipoproteins, which may serve as donors or acceptors of fatty acids [48]
