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

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 hydrolysis of fatty acids than *M. tuberculosis*.

possible enoyl-CoA hydratase), *fadA2* (ML2564, acetyl-CoA-acetyltransferase), *fadB2* (ML2461, 3-hydroxyacyl-CoA dehydrogenase), *fadD19* (ML0352, acyl-CoA synthase), *fadD26* (ML2358, fatty acid-CoA-ligase), *fadD29* (ML0132, probable fatty-acid-CoA synthe‐ tase), *fadD28* (ML0138, possible fatty-acid-CoA synthetase), *fadE25* (ML0737, probable acyl-CoA dehydrogenase) and *fadE5* (ML2563, acyl-CoA dehydrogenase) [59,60]. This gives strong evidence that host lipids provide the main carbon and energy sources for *M. lep‐*

Lipid Inclusions in Mycobacterial Infections http://dx.doi.org/10.5772/54526 41

The *M. leprae* genome contains a gene, coding for an isocitrate lyase, *aceA*. The amino acidsequence of AceA (ML1985c) shows 80 % identity with its homologue from *M. tuberculosis* ICL2 (Rv1915/1916). *AceA* is upregulated in both *M. leprae*-infected nude mouse and human lesions. [59]. A second icl gene, as observed in *M. tuberculosis*, is not present in the genome of *M. leprae*. This finding is of particular interest, because both lyases, *icl* and *icl2*, are jointly required for in vivo growth and virulence [79,80]. Deletion of *icl1* or *icl2* has little effect on bacterial growth in macrophages [80]. So far the *M. leprae* AceA might play a slightly different

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

*rae* during infection.

choesterol esters [10].

role in as the both isocitrate lyases in *M. tuberculosis.*

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

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

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

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 required for mycobacterial persistence.

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, possible enoyl-CoA hydratase), *fadA2* (ML2564, acetyl-CoA-acetyltransferase), *fadB2* (ML2461, 3-hydroxyacyl-CoA dehydrogenase), *fadD19* (ML0352, acyl-CoA synthase), *fadD26* (ML2358, fatty acid-CoA-ligase), *fadD29* (ML0132, probable fatty-acid-CoA synthe‐ tase), *fadD28* (ML0138, possible fatty-acid-CoA synthetase), *fadE25* (ML0737, probable acyl-CoA dehydrogenase) and *fadE5* (ML2563, acyl-CoA dehydrogenase) [59,60]. This gives strong evidence that host lipids provide the main carbon and energy sources for *M. lep‐ rae* during infection.

The *M. leprae* genome contains a gene, coding for an isocitrate lyase, *aceA*. The amino acidsequence of AceA (ML1985c) shows 80 % identity with its homologue from *M. tuberculosis* ICL2 (Rv1915/1916). *AceA* is upregulated in both *M. leprae*-infected nude mouse and human lesions. [59]. A second icl gene, as observed in *M. tuberculosis*, is not present in the genome of *M. leprae*. This finding is of particular interest, because both lyases, *icl* and *icl2*, are jointly required for in vivo growth and virulence [79,80]. Deletion of *icl1* or *icl2* has little effect on bacterial growth in macrophages [80]. So far the *M. leprae* AceA might play a slightly different role in as the both isocitrate lyases in *M. tuberculosis.*
