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

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.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for nearly 2 million deaths each year. The host immune response toward aerosol infection is to quarantine tubercle bacilli in a granulomatous structure (1, 2). However, granuloma-associated mycobacteria can switch to a non-replicative, “dormant” state and successfully evade immune response for decades after infection (3, 4). The metabolic events that permit tubercle bacilli to enter host cells and revive from states of persistence suggest that lipids are utilized as a carbon source (5–7). 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). Upon resuscitation (by the re-introduction of oxygen), these lipid droplets vanish and TAGs are hydrolyzed (11). Unfortunately, the molecular mechanisms for TAG build-up and breakdown are far less well understood in bacteria when compared with those processes in eukaryotes.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). A family of 24 carboxyl ester hydrolases called “lip” genes (lipC to Z, except K and S) has been predicted to play a role in lipid catabolism (14). Among these, only a few have been functionally characterized and related to mycobacterial dormancy and resuscitation (15–18).Tetrahydrolipstatin, a serine esterase inhibitor, covalently binds to and inhibits mammalian lipases and fatty acid synthase (FAS) and is marketed as “Orlistat” for the treatment of severe forms of obesity (19). THL was previously shown to inhibit both active and latent forms of mycobacteria (11, 20–22) but the bacterial target spectrum remains poorly characterized. Therefore, to (1) define the THL target spectrum in a mycobacterial species and (2) to obtain biochemical insights into regulation of lipases and esterases in different metabolic states, we employed a chemical-proteomics approach using activity-based protein profiling (ABPP) with a bait that has been described to bind to lipolytic enzymes (23–25). We identified several known lipases (as anticipated), putative lipase and esterases, and hypothetical proteins of unknown functions, thereby providing a comprehensive resource of experimentally determined THL targets in mycobacteria. Importantly, we systematically compared readouts of fluorescently tagged THL-proteins (7 bands on one-dimensional SDS-PAGE) with those of mass spectrometry-based peptide identification of enriched protein fractions (247 in growing cells). This comparison led to the identification of 14 THL targets, two of which were further validated experimentally. We furthermore provide a conceptual framework for the evaluation of this target list using both experimental as well as bioinformatics approaches in two examples, lipH and tesA. Overall, our data indicate that THL is an anti-mycobacterial drug because of its potential to (1) bind to a relatively wide range of lipolytic enzymes and (2) prevent bacilli from resuscitating from a nonreplicating persistent (NRP) state when lipid metabolism is particularly important.     

The identification of antioxidants in dark soy sauce

Soy sauce is a traditional fermented seasoning in Asian countries, that has high antioxidant activity in vitro and some antioxidant activity in vivo. We attempted to identify the major antioxidants present, using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay as a guide. 3-Hydroxy-2-methyl-4H-pyran-4-one (maltol) was one of several active compounds found in an ethyl acetate extract of dark soy sauce (DSS) and was present at millimolar concentrations in DSS. However, most of the antioxidant activity was present in colored fractions, two of which (CP1 and CP2) were obtained by gel filtration chromatography. Their structural characteristics based on nuclear magnetic resonance (NMR) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) analysis suggest that carbohydrate-containing pigments such as melanoidins are the major contributors to the high antioxidant capacity of DSS.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.