**4. Lipid composition in** *M. leprae* **infected cells**

**3.5. Lipase genes of** *M. leprae*

40 Tuberculosis - Current Issues in Diagnosis and Management

hydrolysis of fatty acids than *M. tuberculosis*.

required for mycobacterial persistence.

**3.6. Enzymes of the β-oxidation and glyoxylate cycle**

the gene might be essential for TAG hydrolysis and growth [69].

In the *M. leprae* genome only 2 lipase genes (*lipG*, *lipU*) were found. But *M. tuberculosis* has also only six expressed Lip enzymes, showing reasonable hydrolase activity for long-chain triacylglycerols. (LipY, LipC, LipL, LipX, LipK, LipG). LipG and LipU from *M. leprae* are homologous with LipG and LipU from *M. tuberculosis* and show sequence identities of 72 and 79%, respectively. The lipases LipG and LipU from *M. tuberculosis* show very low and no activity with long chain triacylglycerols as substrates [47]. *M. tuberculosis* LipY is suspected to be a major functional lipase, which utilizes stored triacylglycerols (TAG) during dormancy and reactivation of the pathogen [47,49]. LipY shows only a weak similarity with *M. leprae* LipU (23 % identity). In summary it appears that *M. leprae* uses different lipases for the

*M. tuberculosis* can grow on fatty acids as sole carbon source and it has been demonstrat‐ ed that fatty acid oxidation is important for survival of the pathogen in the lungs of mice [79,80]. Fatty acids are oxidized via the β-oxidation cycle and the glyoxylate shunt, to re‐ plenish TCA cycle intermediates during growth [81]. The β-oxidation cycle consists of five biochemical reactions, where one molecule acetyl-CoA of the fatty acid is split off per cy‐ cle. The genome of *M. tuberculosis* encodes around 100 genes, designated as fad genes (fat‐ ty acid degradation) with putative roles in the β-oxidation of fatty acids. While *E. coli* has only one enzyme for each step of the β-oxidation cycle, *M. tuberculosis* seems to have sev‐ eral backup enzymes for each reaction [82]. The initial step of β-oxidation is the forma‐ tion of acyl-CoA from free fatty acids and Coenzyme A and is catalyzed by acyl-CoA synthase. In *M. bovis* BCG one Acyl-CoA synthase (BCG1721) (Rv1683) has been identi‐ fied to be exclusively bound to lipid bodies. Nonreplicating mycobacteria, which overex‐ press a BCG1721 construct with an inactive lipase domain displayed a phenotype of attenuated TAG breakdown and regrowth upon resuscitation. These results indicate that

Together with malate synthase, isocitrate lyase (ICL) is the key enzyme of the glyoxylate cycle that catalyzes the cleavage of isocitrate to glyoxylate and succinate [81,83]. The *M. tuberculo‐ sis* genome codes for two isocitrate lyases, icl and icl2, which are essential for the fatty acid metabolism and jointly required for in vivo growth and virulence. Disruption of icl has only little effect on survival in macrophages and bacterial loads in lungs of infected mice. Only disruption of both lyase genes results in a fast elimination of bacteria from lungs of infected mice and infected macrophages [79,80]. These results strongly suggest that both icl genes are

All enzymes involved in lipid metabolism in lipid bodies are summarized in Table 1.

*M. leprae* has approximately one-third as many potential fad enzymes with probable roles in the β-oxidation. Even though *M. leprae* genome contains less necessary β-oxidation cy‐ cle genes than *M. tuberculosis*, transcript analysis revealed expression of acyl-CoA meta‐ bolic enzymes including *echA1* (ML0120, putative enoyl-CoA hydratase), *echA12* (ML1241, In 1863, Virchow described foamy cells, which form droplets and surround *M. leprae* within the phagolysomes. [84,85]. This lipid capsule forms a characteristic electron-transparent zone. In contrast to *M. tuberculosis*, the presence of lipid bodies seem to be rather exceptional in *M. leprae* [85]. The lipid capsule contains mycoserosoic acids of phthiocerol dimycocerosates as well as phenolic glycolipids [86,87]. Brennan reported the full characterization of three phenolphthiocerol triglycosides by *M. leprae* [84]. It has been postulated that many of these molecules together with phosphatidylinositol mannosides and phospholipids are released from the cell wall after synthesis, forming the capsule-like region [11]. The dominant lipid in the cell wall which gives *M. leprae* immunological specificity is phenolic glycolipid-1 (PGL-1). Phenolic glycolipid 1 has been isolated in relatively high concentrations from purified bacteria and from *M. leprae* infected tissues [88]. PGL-1 is thought to be a major component of the capsule in *M. leprae* and constitutes an important interface between bacteria and host [89]. It has been suggested that PGL-1 is involved in the interaction of *M. leprae* with the laminin of Schwann cells, thus PGL-1 hight play a role in peripheral nerve-bacillus interactions [90]. Moreover, phenolic glycolipids seem to be involved in the in the stimulation of suppressor T-cells in lepromatous leprosy [91]. Recently it was reported that also LDs from *M. leprae* infected SCs and macrophages accumulate mainly host derived lipids, such as oxidized phospholipids [92]. BODIPY stains infected SCs, indicating that LDs contain neutral lipids, such as triacylglycerols (TAG), but it seems as *M. leprae*-infected cells accumulate large amounts of cholesterol and choesterol esters [10].


**5. Induction of lipid droplet biogenesis**

formation and development of lipid droplets.

cytokine response [100].

[104] (Figure 3).

**5.2. TLR mediated LD formation in** *M. bovis* **and** *M. leprae*

Since the biogenesis of lipid droplets in macrophages seems an absolute requirement for intracellular bacteria to establish infections, we will discuss mechanisms involved in foam cell

Lipid Inclusions in Mycobacterial Infections http://dx.doi.org/10.5772/54526 43

Upon infection with pathogenic bacteria macrophages generate reactive oxygen species (ROS). The release of ROS generates oxidative stress, and results not only in damage to cellular structures but also to oxidation of fatty acids, such as low density lipoproteins (OxLDL) in granulomas. The binding of OxLDL to type 1 scavenger receptors CD36 and LOX1 induces increased surface expression of both receptors, leading to uptake of OxLDL [96-98]. In addition, CD36 increases the uptake of *M. tuberculosis* by macrophages [99]. The increased rate of OxLDL uptake results in the accumulation of oxidized lipids, which finally leads to the formation of foamy macrophages [98]. *M. tuberculosis* and *M. leprae* benefit from the accumulated OxLDL in the infected macrophage. OxLDL-laden lung macrophages show enhanced replication of intracellular *M. tuberculosis* compared to macrophages loaded with non-oxidized LDL [98]. The presence of oxidized phospholipids in *M. leprae* infected macrophages down-regulates the innate immune response and contributes to pathogenesis [92]. Moreover, scavenger receptordeficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower

*Mycobacterium bovis* Bacillus Calmette-Guérin (BCG) and *M. leprae* are recognised by the Tolllike receptors (TLR) TLR6 and TLR2 [101,102]. Mycobacterium bovis Bacillus Calmette-Guérin induced lipid body formation is TLR2 mediated [103]. The mycobacterial surface molecule lipoarabinomannan (LAM) induces the formation of foamy macrophages by binding to TLR2

*M. leprae* association to macrophages is mediated by binding of the bacteria to TLR2 and TLR6. Heterodimerization of TLR2 and TLR6 leads to downstream signalling and subsequent LD formation [102,105]. Macrophage association is not dependent on binding to TLR2 or TLR6. Neither a TLR2-/- or TLR6-/- knockout macrophage shows reduced binding to *M. leprae*. This suggests that both TLR2 and TLR6 can bind *M. leprae* alone, or/and the presence of other receptors, binding to *M. leprae*. The TLR2-/- or TLR6-/- knockout macrophages do also not completely abolish LD formation, but show only reduced LD formation [102]. This suggests the presence of additional signalling pathways for LD formation. In SCs TLR6, but not TLR2, is essential for *M. leprae*-induced LD biogenesis in [101]. In LL lesions, accumulated with LD enriched macropages the genes for ADRP and CD36 are up-regulated [30,92,102]. This suggests

also an involvement of CD36 in LD formation of *M. leprae* (Figure 4) [99].

**5.1. Scavenger receptor mediated lipid droplet biogenesis in** *M. tuberculosis*

**Table 1.** Enzymes involved in lipid body metabolism in *M. tuberculosis* and *M. bovis* BCG. Homologous genes in *M. tuberculosis* H37Rv are written in square brackets. NA, not applicable. \*, expressed in yeast as recombinant protein. DGAT, diacylglycerol acyltransferase
