Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids

Abstract

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

Reversible Lipid Accumulation and Associated Division Arrest of Mycobacterium avium in Lipoprotein-Induced Foamy Macrophages May Resemble Key Events during Latency and Reactivation of Tuberculosis

“…After cleavage of its PE domain, LipY was seen to be inserted in the outermost layer of the cell wall (53,54). At present, the possibility that other lipases, such as cutinase (61,66) or monoglyceride lipases (67,68), play a role in host TAG hydrolysis cannot be excluded.…”

Triacylglycerol Utilization Is Required for Regrowth of In Vitro Hypoxic Nonreplicating Mycobacterium bovis Bacillus Calmette-Guerin

“…This drug acts as an irreversible inhibitor of pancreatic and gastric lipases (5, 13). THL has also been shown to have bactericidal effects on some mycobacteria (14) and some activity against an extracellular lipase encoded by Rv0183 in M. tuberculosis (7).…”

“…Genes encoding approximately 24 TG lipases have been predicted to be present in the genome of M. tuberculosis (9), and the lipY (Rv3097c) and Rv0183 products have been experimentally shown to possess TG lipase activity (7,9). THL, an inhibitor of pancreatic lipases, reduced total lipase activity and prevented breakdown of TGs and regrowth of mycobacteria when they were resuscitated with oxygen ( Fig.…”

A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

“…E. coli Rosetta(DE3)/ pLysS cells were transformed with the pDest14-His 6 -MSMEG_0220 plasmid. MSMEG_0220 was produced and purified as previously described in the case of Rv0183, the homologue of MSMEG_0220 from M. tuberculosis (7). Briefly, the cell pellet recovered from TB cell cultures (1 liter) was resuspended in ice-cold lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.1% [vol/vol] Triton X-100, 0.25 mg ml Ϫ1 lysozyme; 30 ml per initial liter of culture).…”