Abstract
Mycobacterium tuberculosis Rv3775 (LipE) was annotated as a putative lipase. However, its lipase activity has never been characterized, and its precise role in tuberculosis (TB) pathogenesis has not been thoroughly studied to date. We overexpressed and purified the recombinant LipE (rLipE) protein and demonstrated that LipE has a lipase/esterase activity. rLipE prefers medium-chain ester substrates, with the maximal activity on hexanoate. Its activity is the highest at 40°C and pH 9. We determined that rLipE hydrolyzes trioctanoate. Using site-directed mutagenesis, we confirmed that the predicted putative activity triad residues Ser97, Gly342, and His363 are essential for the lipase activity of rLipE. The expression of the lipE gene was induced under stressed conditions mimicking M. tuberculosis' intracellular niche. The gene-disrupting mutation of lipE led to significantly reduced bacterial growth inside THP-1 cells and human peripheral blood mononuclear cell-derived macrophages and attenuated M. tuberculosis infection in mice (with ∼8-fold bacterial load reduction in mouse lungs). Our data suggest that LipE functions as a lipase and is important for M. tuberculosis intracellular growth and in vivo infection.
Highlights
Mycobacterium tuberculosis Rv3775 (LipE) was annotated as a putative lipase
The sequence alignment revealed that LipE has an SxxK motif at aa 97 to 100, which is conserved in LipD, LipL, LipP, and EstA of C. crescentus (Fig. 1A)
The results showed that the EstA (PDB 5GKV.1) from C. crescentus had the highest similarity with the LipE sequence (41 identical amino acids, ranging from aa 26 to 405), and the 3D structure model of LipE using the EstA as a template had the lowest root mean square deviation (RMSD) value (RMSD ϭ 0.144)
Summary
Mycobacterium tuberculosis Rv3775 (LipE) was annotated as a putative lipase. its lipase activity has never been characterized, and its precise role in tuberculosis (TB) pathogenesis has not been thoroughly studied to date. Our data suggest that LipE functions as a lipase and is important for M. tuberculosis intracellular growth and in vivo infection. Some proteins involved in lipid metabolism of M. tuberculosis are virulence related, and mutations of them lead to attenuated phenotypes in cell and animal infection. Bolism is an important requirement for M. tuberculosis infection and persistence in hosts, functional characterization of the specific lipases/esterases in M. tuberculosis lipid/ester catabolism pathways provides an opportunity to discover new mechanisms of tuberculosis (TB) pathogenesis. We characterized the lipase/esterase activity of recombinant LipE (rLipE) and evaluated its catalytic triad and its hydrolysis of triglycerides. We defined the impact of LipE on M. tuberculosis intracellular growth in macrophages and on M. tuberculosis in vivo infection
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