Triacylglycerol Utilization Is Required for Regrowth of In Vitro Hypoxic Nonreplicating Mycobacterium bovis Bacillus Calmette-Guerin

Mycobacteria store triacylglycerols (TGs) under various stress conditions, such as hypoxia, exposure to nitric oxide, and acidic environments. These stress conditions are known to induce nonreplicating persistence in mycobacteria. The importance of TG accumulation and utilization during regrowth is not clearly understood. Here we specifically determined the levels of accumulated TG and TG lipase activity in Mycobacterium bovis bacillus Calmette-Guerin (BCG) in various different physiological states (logarithmic growth, aerated stationary phase, hypoxia-induced dormancy, and regrowth from dormancy). We found extensive accumulation and degradation of TGs in the bacilli during entry into and exit from hypoxia-induced dormancy, respectively. These processes are accompanied by dynamic appearance and disappearance of intracellular TG lipid particles. The reduction in TG levels coincides with an increase in cellular TG lipase activity in the regrowing bacilli. Tetrahydrolipstatin, an inhibitor of TG lipases, reduces total lipase activity, prevents breakdown of TGs, and blocks the growth of mycobacteria upon resuscitation with air. Our results demonstrate that utilization of TGs is essential for the regrowth of mycobacteria during their exit from the hypoxic nonreplicating state.Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), one of the major infectious diseases, which affects one-third of the world''s population (http://www.who.int/mediacentre/factsheets/fs104/en/). The majority of TB patients carry a latent infection. However, reactivation leading to active disease (16, 27) often occurs once the host defenses are weakened. During the latency period, mycobacteria are tolerant to many conventional antibiotics (23, 28), thus making eradication of TB challenging.In the human body, M. tuberculosis is believed to persist in lung lesions with hypoxic environments (6, 27). Wayne and Hayes established an “in vitro dormancy model” in which mycobacterial cultures are subjected to gradual depletion of oxygen, which causes the obligate aerobic cells to exit the cell cycle and enter into a nonreplicating persistent state (26). The bacilli in the nonreplicating persistent state are phenotypically drug resistant. Recent efforts to explore the mechanisms which allow the tubercle bacilli to enter into dormancy and survive in the host for a long period of time suggest that fatty acids (FAs) could be an important source of energy during the persistent state (1, 17, 20). In particular, triacylglycerols (TGs), a class of neutral lipids, are postulated to be a likely source of FAs (8). TGs are an efficient form of energy reserves in many organisms during long-term survival.Recently, it was reported that tubercle bacilli in sputum from patients with TB contain lipid bodies (11). Moreover, TGs accumulate in hypervirulent W-Beijing strains of M. tuberculosis (19). It is interesting that TGs accumulate in these clinical strains of TB. However, no systematic study has been carried out yet to investigate the accumulation and degradation of TG species under various physiological conditions in mycobacteria. Here we used M. bovis bacillus Calmette-Guerin (BCG) to study the kinetics of TG buildup and breakdown during different growth phases, including logarithmic growth, aerated stationary phase, hypoxia-induced dormancy (Wayne model), and regrowth from dormancy upon reaeration of hypoxic cultures. Utilizing tetrahydrolipstatin (THL) as a chemical probe, we showed that mobilization of TGs is essential for the regrowth of mycobacteria during recovery from the hypoxia-induced dormant state.     

Spin distribution of the H-cluster in the Hox–CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of 14N and 13C nuclear interactions

Hydrogenases are enzymes which catalyze the reversible cleavage of molecular hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the “H-cluster.” It consists of the redox-active binuclear subcluster ([2Fe]_H) coordinated by CN⁻ and CO ligands and the cubane-like [4Fe–4S]_H subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe]_H subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the H_ox–CO state the open coordination site at the [2Fe]_H subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepared with ¹³CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three ¹³C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were observed and assigned to ¹³CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, respectively. No ¹³CO exchange of the CO ligand to the proximal iron was observed. The hyperfine interactions detected indicate a rather large distribution of the spin density over the terminal and bridging CO ligands attached to the distal iron. Furthermore, ¹⁴N nuclear spin interactions were measured. On the basis of the observed ¹⁴N hyperfine couplings, which result from the CN⁻ ligands of the [2Fe]_H subcluster, it has been concluded that there is very little unpaired spin density on the cyanides of the binuclear subcluster.

Characterization of Substrate Preference for Slc1p and Cst26p in Saccharomyces cerevisiae Using Lipidomic Approaches and an LPAAT Activity Assay

Background

Phosphatidic acid (PA) is a key regulated intermediate and precursor for de novo biosynthesis of all glycerophospholipids. PA can be synthesized through the acylation of lysophosphatidic acid (LPA) by 1-acyl-3-phosphate acyltransferase (also called lysophosphatidic acid acyltransferase, LPAAT). Recent findings have substantiated the essential roles of acyltransferases in various biological functions.

Methodologies/Principal Findings

We used a flow-injection-based lipidomic approach with ∼200 multiple reaction monitoring (MRM) transitions to pre-screen fatty acyl composition of phospholipids in the yeast Saccharomyces cerevisiae mutants. Dramatic changes were observed in fatty acyl composition in some yeast mutants including Slc1p, a well-characterized LPAAT, and Cst26p, a recently characterized phosphatidylinositol stearoyl incorporating 1 protein and putative LPAAT in S. cerevisiae. A comprehensive high-performance liquid chromatography–based multi-stage MRM approach (more than 500 MRM transitions) was developed and further applied to quantify individual phospholipids in both strains to confirm these changes. Our data suggest potential fatty acyl substrates as well as fatty acyls that compensate for defects in both Cst26p and Slc1p mutants. These results were consistent with those from a non-radioactive LPAAT enzymatic assay using C17-LPA and acyl-CoA donors as substrates.

Conclusions

We found that Slc1p utilized fatty acid (FA) 18:1 and FA 14:0 as substrates to synthesize corresponding PAs; moreover, it was probably the only acyltransferase responsible for acylation of saturated short-chain fatty acyls (12:0 and 10:0) in S. cerevisiae. We also identified FA 18:0, FA 16:0, FA 14:0 and exogenous FA 17:0 as preferred substrates for Cst26p because transformation with a GFP-tagged CST26 restored the phospholipid profile of a CST26 mutant. Our current findings expand the enzymes and existing scope of acyl-CoA donors for glycerophospholipid biosynthesis.     

Fast and reliable determination of the antifungal drug voriconazole in plasma using monolithic silica rod liquid chromatography

In the present study, we developed a fast and reliable HPLC assay for the determination of the new triazole antifungal agent voriconazole in plasma, using a Chromolith RP 18e (100 mm x 4.6 mm) monolithic silica rod HPLC column. After liquid-liquid extraction, plasma samples were separated with a mobile phase consisting of ammoniumdihydrogencarbonate buffer (pH 5.8)-acetonitrile-tetrahydrofuran (72:25:3) at a flow-rate of 3.5 mL/min and UV detection at 255 nm. The retention times for voriconazole and internal standard (UK-115794) were 2.3 and 2.7 min, respectively, and total run time was 4 min. The calibration curves were linear between 0.05 and 10 microg/mL, and within-assay and between-assay coefficients of variation were <4%. The proposed assay for voriconazole in plasma is fast, sensitive and reliable, and, thus, well suited for routine therapeutic monitoring of patients and for pharmacokinetic studies. It can be predicted that the use of monolithic silica rod chromatography will substantially shorten the turn-around time in the therapeutic drug monitoring laboratory.     

The domains of protein S from Myxococcus xanthus: structure, stability and interactions

Protein S from Myxococcus xanthus is a member of the beta gamma-crystallin superfamily. Its N and C-terminal domains (NPS and CPS, respectively) show a high degree of structural similarity and possess the capacity to bind two calcium ions per domain. For NPS, their positions were determined by X-ray diffraction at 1.8 A resolution, making use of molecular replacement with the NMR structure as search model. The overall topology of NPS is found to be practically the same as in complete protein S. In natural protein S, the domains fold independently, with a significant increase in stability and cooperativity of folding in the presence of Ca2+. The recombinant isolated domains are stable monomers which do not show any tendency to combine to "nicked" full-length protein S. In order to investigate the stability and folding of natural protein S and its isolated domains, spectroscopic techniques were applied, measuring the reversible urea and temperature-induced unfolding transitions at varying pH. The increment of Ca2+ to the free energy of stabilization amounts to -10 and -5 kJ/mol for NPS and CPS, respectively. For both NPS and CPS, in the absence and in the presence of 3 mM CaCl2, the two-state model is valid. Comparing DeltaGU-->N for CPS (-21 kJ/mol at pH 7, liganded with Ca2+) with its increment in the intact two-domain protein, the stability of the isolated domain turns out to be decreased in a pH-dependent manner. In contrast, the stability of Ca2+-loaded NPS (DeltaGU-->N=-31 kJ/mol, pH 7) is nearly unchanged down to pH 2 where Ca2+ is released (DeltaGU-->N=-26 kJ/mol, pH 2). In intact protein S, the N-terminal domain is destabilized relative to NPS. Evidently, apart from Ca2+ binding, well-defined domain interactions contribute significantly to the overall stability of intact protein S.     

Rapid atrazine mineralisation in soil slurry and moist soil by inoculation of an atrazine-degrading Pseudomonas sp. strain

The evaluation of pesticide-mineralising microorganisms to clean-up contaminated soils was studied with the widely applied and easily detectable compound atrazine, which is rapidly mineralised by several microorganisms including the Pseudomonas sp. strain Yaya 6. The rate of atrazine removal was proportional to the water content of the soil and the amount of bacteria added to the soil. In soil slurry, 6 mg atrazine kg soil⁻¹ was eliminated within 1 day after application of 0.3 g dry weight inoculant biomass kg soil⁻¹ and within 5 days when 0.003 g kg soil⁻¹ was used. In partially saturated soil (60% of the maximal water-holding capacity) 15 mg atrazine kg soil⁻¹ was eliminated within 2 days by 1 g biomass kg soil⁻¹ and within 25 days when 0.01 g biomass kg soil⁻¹ was used. In unsaturated soil, about 60% [U-ring-¹⁴C]atrazine was converted to ¹⁴CO₂ within 14 days. Atrazine was very efficiently removed by the inoculant biomass, not only in soil that was freshly contaminated but also in soil aged with atrazine for up to 260 days. The bacteria exposed to atrazine in unsaturated sterile soil were still active after a starvation period of 240 days: 15 mg newly added atrazine kg soil⁻¹ was eliminated within 5 days. Received: 31 October 1997 / Received revision: 16 January 1998 / Accepted: 18 January 1998     

Structural and biochemical characterization of the type III secretion chaperones CesT and SigE.

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir-CesT and enzymatic activity profiles of SigD-SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

A contribution to the generalization of the Gompertz function

SCHARF (1976) discusses various growth models. For the Gompertz function the differential equation (Formula: see text) is used. In words: the difference between relative growth rate and relative growth acceleration is constant. On the other hand, according to WENK (1973), the differential equation (Formula: see text) applies to the Gompertz function. It can be shown mathematically that (Formula: see text) applies in general. From Eq. (2) one obtains without trouble (Formula: see text). Therefore, the property leading to the Gompertz function may be defined as follows; the logarithmic derivation of the relative growth rate is constant. Eq. (2) is applicable only in special cases. It can be extended by assuming that c is not constant, but a function of time. In this way, a great number of growth functions can be found, which have to be regarded as model-based extensions of the Gompertz function.