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

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‐

38 Tuberculosis - Current Issues in Diagnosis and Management

zymes are involved in TAG synthesis during latency and pathogenicity [69].

active. [18,70].

maintenance.

host cell [71].

lesions [59].

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

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

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

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 Neutral lipids in the core of the lipid body are hydrolyzed by lipases or esterases, yielding fatty acids for energy generation and anabolism of membrane phospholipids.

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

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 it may hydrolyze host's TAG [75].

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 TAG hydrolysis.
