Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Madhu Sudhan Ravindran,Srinivasa P.S Rao,Xiamin Cheng,Ankit Shukla,Amaury Cazenave-Gassiot,Shao Q Yao,Markus R Wenk

doi:10.1074/mcp.m113.029942

Abstract

Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/β-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.

Highlights

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for nearly 2 million deaths each year
Cells were harvested from these three conditions, washed and lysed (Fig. 1A, step 1) and THL probes were incubated with total cell lysates to allow binding with proteins (Fig. 1A, step 2)
THL bound within the LipH active site (Fig. 4B) with a binding energy of Ϫ7.10 Kcal/mol, which was comparable to Ϫ5.11 Kcal/mol of THL with virtually identical orientation of the catalytic triad (Fig. 4C). Using this approach for TAGs with increasing fatty acyl chain lengths, we found that species with fatty acids (FAs) carbon chain lengths of 6 – 8 are predicted to be energetically preferred substrates whereas chain lengths Ͼ10 do not lead to any docking (Fig. 4D); this is consistent with the biochemical results with synthetic substrates (Fig. 3E, 3F) [39, 46]

Summary

Introduction

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for nearly 2 million deaths each year. During times of oxygen deprivation and in the absence of host cells, cultivated mycobacteria store fatty acids (FAs) in the form of triacylglycerol (TAG)1-enriched lipid droplets (8 –10). Comparative sequence analysis of the Mtb genome has revealed that it contains 250 genes encoding enzymes involved in lipid metabolism compared with only 50 enzymes in Escherichia coli, which has a genome of comparable size. Among these genes, 150 are predicted to encode proteins involved in lipid catabolism [12, 13]. THL was previously shown to inhibit both active and latent forms of mycobacteria (11, 20 – 22) but the bacterial target spectrum remains poorly characterized. To [1] define the THL target spectrum in a mycobacterial species and [2] to obtain biochemical insights

Objectives

Results

Conclusion