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.

Figure 1. Amino acid sequence alignment between Rv0183, without its putative signal peptide identified from the bioinformatic results, and three homologous proteins from M. bovis (Mb0189, 100%), M. leprae (ML2603, 79%) and M. smegmatis (68%), and a human (34%) and a mouse (36%) MGL

This figure was generated by the ESPrit program (available from the Expasy web site) using the alignment function performed by Clustal W [from the EBI (European Bioinformatics Institute) web site] and the secondary element structure obtained from the PredictProtein website [50]. Conserved residues are shown with a black background with similar residues in grey. Residues potentially involved in the catalytic triad are indicated by a triangle (▲). The two lipase motifs are indicated by circles (●). The secondary structure elements (β-strands and α-helices) are indicated at the top.

Figure 2. SDS/PAGE of the purification and the TEV cleavage of rRv0183

Protein samples were loaded onto a SDS/PAGE (12% gel) under reducing conditions. The gel was stained with Coomassie brilliant blue R250. Lane 1, molecular mass markers from Amersham Biosciences; lane 2, purified rRv0183 (10 μg); lane 3, purified rRv0183 after TEV cleavage and after exclusion from Ni²⁺-NTA agarose (10 μg).

Figure 3. TLC analysis of triolein degradation by HPL and rRv0183

The reaction was performed on triolein emulsion with HPL (140 μg) for 1 h and rRv0183 (780 μg) was then added to the mixture containing the HPL degradation products. Lipids were extracted with a chloroform/methanol solution and separated on silica gel by TLC (see Materials and methods section). Lane 1, reaction mixture before incubation with enzymes; lane 2, products released after 1 h of incubation with HPL; lanes 3, 4 and 5, products released 10, 20 and 120 min respectively after adding rRv0183; lane 6, lipid markers. FA, freeoleic acid.

Figure 4. Lipolytic activity of rRv0183 on DiC₁₀, mono-olein, diolein and purified soybean oil monolayers as a function of the surface pressure

Lipolytic activity was determined after injecting 15 μg of rRv0183 into the reaction compartment. Collapse point of each substrate was reached at 41, 32, 28 and 13.8 mN·m⁻¹ in the case of DiC₁₀, mono-olein, diolein and purified soybean oil respectively. Each experiment was performed in triplicate (error ±5%).

Figure 5. Kinetic assays on rRv0183 using a mono-olein substrate

(A) pH stability, (B) pH effects on rRv0183 activity, (C) temperature dependence and (D) NaTDC dependence (for details of the experimental procedure, see Materials and methods section).

Figure 6. Inhibitory effects of E600 (○), THL (◇) and PMSF (□) on rRv0183 activity as a function of time

(A) Without NaTDC and (B) with 3 mM NaTDC. The protein/inhibitor ratio was 1:200 in all experiments. Enzyme activity was determined as described in the Materials and methods section.

Figure 7. Location of Rv0183 in M. tuberculosis cellular compartments

Aliquots (15 μg of proteins per well) of M. tuberculosis cellular compartments were subjected to immunoblotting analysis using purified polyclonal antibodies directed against rRv0183. Lane 1, rRv0183 purified protein (400 ng); lane 2, protein from culture medium obtained using a 0.22 μm filter; lane 3, protein from culture medium; lane 4, cell wall; lane 5, membrane; lane 6, Triton X-114 extracted proteins; lane 7, cytosol. M. tuberculosis cellular samples were kindly provided by the TB (tuberculosis) Vaccine Testing and Research Materials funded by NIH and NIAID (Colorado State University).