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

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-Gué rin cultured under replicating and non-replicating conditions. THL targets ␣/␤-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. Molecular & Cellular

Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria

“…As expected for a drug synergizing with vancomycin against M. tuberculosis and M. bovis BCG growth, we observed a reduction of PDIM A and PDIM B in THL-treated M. tuberculosis. The interaction of THL with TesA might explain, at least partly, this result (20). In agreement with Belardinelli et al, we also observed that the outer membrane SL-1 lipids were strongly decreased (37).…”

“…We also observed a reduction of TAG. Altogether, our results suggest that THL induced a loss of cell wall integrity by targeting several cell envelope lipid pathways, potentially including Chp 1, involved in SL-1 synthesis as previously reported (30), and TesA, involved in PDIM synthesis, as suggested by Ravindran et al (20). Simvastatin, although not able to synergize with vancomycin, inhibited already alone (100 g/ml) the growth of both M. bovis BCG and M. tuberculosis.…”

“…Parker et al reported that, in M. smegmatis, THL can target Rv3802, acting both as a thioesterase and as a phospholipase on phosphatidylinositol mannoside 2 (18) but also the ES-31 serine protease (19). Interestingly, Ravindran et al pointed out nine lipases and one thioesterase (TesA) as potential THL targets (20). This last enzyme, TesA, is involved in the synthesis of phthiocerol dimycocerosate (PDIM) (21).…”

An Antibacterial β‐Lactone Kills Mycobacterium tuberculosis by Disrupting Mycolic Acid Biosynthesis

“…[15] b-Lactones are attractive molecules for this endeavor since their substitution pattern is reminiscent of the signature mycolic acid b-hydroxy motif,f ormed by ring opening and covalent active-site acylation( Figure 1C). Previous studies with as mall number of b-lactones in mycobacteria revealed targeting of diverse lipases [16] as well as two caseinolytic proteases termed ClpP1 and ClpP2. [17] Here we profile apanel of b-lactones for anti-mycobacterial activity and identify one hit compound with bactericidal activity.I n-depth mode-of-action analysis reveals Pks13 and Ag85 enzymes as predominant targets.…”

Ein antibakterielles β‐Lacton bekämpft Mycobacterium tuberculosis durch Infiltration der Mykolsäurebiosynthese

“…[15] b-Lactone sind fürd iese Rolle überaus vielversprechende Bausteine,d ai hre Konfiguration der von Mykolsäuren angepasst werden kann und sie bei einer Bindung an ein Serin des aktiven Zentrums nach der Ringçffnung das gleiche bHydroxysäuremotiv aufweisen (Abbildung 1C). Studien mit einer Anzahl an b-Lactonen in Mykobakterien haben sowohl diverse Lipasen [16] als auch zwei caseinolytische Proteasen, ClpP1 und ClpP2, [17] als molekulare Ziele ausgemacht. Hier testen wir b-Lactone auf antimykobakterielle Aktivitätu nd identifizieren eine bakteriolytische Verbindung,d eren detaillierte Analyse Pks13 und Ag85 als vorrangige Zielproteine offenlegt.…”