Phosphatidylcholine Synthesis Research Articles

Cytidine 5'-Diphosphocholine Corrects Alveolar Type II Cell Mitochondrial Dysfunction in Influenza-infected Mice.

Development of acute respiratory distress syndrome (ARDS) in influenza A virus (IAV)-infected mice is associated with inhibition of ATII (alveolar type II) epithelial cell de novo phosphatidylcholine synthesis, and administration of the phosphatidylcholine precursor cytidine 5'-diphosphocholine (CDP-choline) attenuates IAV-induced acute respiratory distress syndrome in mice. We hypothesized inhibition of phosphatidylcholine synthesis would also impact the function of ATII cell mitochondria. To test this hypothesis, adult C57BL/6 mice of both sexes were inoculated intranasally with 10,000 pfu/mouse influenza A/WSN/33 (H1N1). Control mice were mock-infected with virus diluent. Mice were treated with saline vehicle or CDP-choline (100 μg/mouse i.p.) once daily from 1 to 5 days postinoculation (dpi). ATII cells were isolated by a standard lung digestion protocol at 6 dpi for analysis of mitochondrial function. IAV infection increased uptake of the glucose analog fludeoxyglucose F 18 by the lungs and caused a switch from oxidative phosphorylation to aerobic glycolysis as a primary means of ATII cell ATP synthesis by 6 dpi. Infection also induced ATII cell mitochondrial depolarization and shrinkage, upregulation of PGC-1α, decreased cardiolipin content, and reduced expression of mitofusin 1, OPA1, DRP1, complexes I and IV of the electron transport chain, and enzymes involved in cardiolipin synthesis. Daily CDP-choline treatment prevented the declines in oxidative phosphorylation, mitochondrial membrane potential, and cardiolipin synthesis resulting from IAV infection but did not fully reverse the glycolytic shift. CDP-choline also did not prevent the alterations in mitochondrial protein expression resulting from infection. Taken together, our data show ATII cell mitochondrial dysfunction after IAV infection results from impaired de novo phospholipid synthesis, but the glycolytic shift does not.

Reciprocal regulation of phosphatidylcholine synthesis and H3K36 methylation programs metabolic adaptation.

Phospholipid biosynthesis plays a role in mediating membrane-to-histone communication that influences metabolic decisions. Upon nutrient deprivation, phospholipid methylation generates a starvation signal in the form of S-adenosylmethionine (SAM) depletion, leading to dynamic changes in histone methylation. Here we show that the SAM-responsive methylation of H3K36 is critical for metabolic adaptation to nutrient starvation in the budding yeast Saccharomyces cerevisiae. We find that mutants deficient in H3K36 methylation exhibit defects in membrane integrity and pyrimidine metabolism and lose viability quickly under starvation. Adjusting the synthesis of phospholipids potently rewires metabolic pathways for nucleotide synthesis and boosts the production of antioxidants, ameliorating the defects resulting from the loss of H3K36 methylation. We further demonstrate that H3K36 methylation reciprocally regulates phospholipid synthesis by influencing redox balance. Our study illustrates an adaptive mechanism whereby phospholipid synthesis entails a histone modification to reprogram metabolism for adaptation in a eukaryotic model organism.

Mechanism of action and therapeutic route for a muscular dystrophy caused by a genetic defect in lipid metabolism

CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial β-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb−/− affected muscle. Treatment of Chkb−/− myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for β-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury.

Tormentic Acid Ameliorates Hepatic Fibrosis in vivo by Inhibiting Glycerophospholipids Metabolism and PI3K/Akt/mTOR and NF-κB Pathways: Based on Transcriptomics and Metabolomics.

This study aimed to investigate the effects and underlying mechanisms of tormentic acid (TA) on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. The rats were intragastrically administered with 50% CCl4 for 9 weeks to induce hepatic fibrosis, followed by various agents for 6 weeks. Transcriptomic analysis was carried out to predict the potential targets, and then multiple examinations were performed to verify the prediction. The results showed that TA significantly alleviated liver injury and fibrosis, as evidenced by the ameliorative pathological tissue, low transaminase activity, and decreased collagen accumulation. Besides, TA markedly reduced hepatocyte apoptosis by regulating the expression of caspase-3 and Bcl-2 families. The transcriptomic analysis revealed 2,173 differentially expressed genes (DEGs) between the TA and model groups, which could be enriched in the metabolic pathways and the PI3K/Akt and NF-κB signaling pathways. The metabolomics analysis showed that TA could regulate the glycerophospholipid metabolism pathway by regulating the synthesis of phosphatidylserines, phosphatidylethanolamines and phosphatidylcholines. Moreover, the integrative analysis of the transcriptomics and metabolomics data indicated that TA inhibited the glycerophospholipid metabolism pathway by inhibiting the expression of LPCAT4, PTDSS2, PLA2G2A and CEPT1. In addition, the relevant signaling pathways analysis confirmed that TA inhibited HSCs activation by blocking the PI3K/Akt/mTOR pathway and ameliorated inflammatory injury by inhibiting the NF-κB pathway. In conclusion, TA significantly alleviates liver fibrosis in vivo by inhibiting the glycerophospholipid metabolism pathway and the PI3K/Akt/mTOR and NF-κB signaling pathways.

Perspective: Estrogen and the Risk of Cognitive Decline: A Missing Choline(rgic) Link?

ABSTRACTFactors that influence the risk of neurocognitive decline and Alzheimer's disease (AD) may provide insight into therapies for both disease treatment and prevention. Although age is the most striking risk factor for AD, it is notable that the prevalence of AD is higher in women, representing two-thirds of cases. To explore potential underlying biological underpinnings of this observation, the intent of this article is to explore the interplay between cognitive aging and sex hormones, the cholinergic system, and novel hypotheses related to the essential nutrient choline. Mechanistic evidence points toward estrogen's neuroprotective effects being strongly dependent on its interactions with the cholinergic system, a modulator of attentional functioning, learning, and memory. Estrogen has been shown to attenuate anticholinergic-induced impairments in verbal memory and normalize patterns of frontal and occipital cortex activation, resulting in a more “young adult” phenotype. However, similar to estrogen replacement's effect in cardiovascular diseases, its putative protective effects may be restricted to early postmenopausal women only, a finding supportive of the “critical window hypothesis.” Estrogen's impact on the cholinergic system may act both locally in the brain but also through peripheral tissues. Estrogen is critical for inducing endogenous choline synthesis via the phosphatidylethanolamine N-methyltransferase (PEMT) gene–mediated pathway of phosphatidylcholine (PC) synthesis. PEMT is dramatically induced in response to estrogen, producing not only a PC molecule and source of choline for the brain but also a key source of the long-chain ω-3 fatty acid, DHA. Herein, we highlight novel hypotheses related to hormone replacement therapy and nutrient metabolism aimed at directing future preclinical and clinical investigation.

The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids

Legionella micdadei is responsible for community- or nosocomial-acquired pneumonia as well as the influenza-like illness Pontiac fever. The aim of this study was to investigate the ability of L. micdadei to utilize extracellular choline for phosphatidylcholine (PC) synthesis and its consequences for the phospholipid composition of its membrane system and the interaction with the human LL-37 peptide. Comparative analysis of the PC content using isotopic labeling revealed that in presence of exogenous choline 98% of the total PC was synthesized via the Pcs pathway while the remaining 2% were generated via the PE-methylation (PmtA) pathway. PC species were to a greater extent defined by the Pcs pathway in the outer membrane than in the inner membrane. While no major changes in the bacterial lipid content were observed using 31P NMR, indication for utilization of longer acyl chains and slight increase of PG in response to choline addition was observed by a top-down lipidomics screen. The LL-37 peptide inhibited L. micdadei growth in a dose-dependent manner. Bacteria cultured with exogenous choline were more sensitive to the LL-37 peptide when compared to the standard culture condition. Our biophysical investigations show that the peptide perturbs bacterial-derived phospholipid monolayers and this interaction is dependent on the molar portion of PC. This interaction is responsible for the observed changes in the anti-L. micdadei activity of the LL-37 peptide.

Bi-allelic variants in CHKA cause a neurodevelopmental disorder with epilepsy and microcephaly.

The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.

Rewiring phospholipid biosynthesis reveals resilience to membrane perturbations and uncovers regulators of lipid homeostasis

The organelles of eukaryotic cells differ in their membrane lipid composition. This heterogeneity is achieved by the localization of lipid synthesizing and modifying enzymes to specific compartments, as well as by intracellular lipid transport that utilizes vesicular and non‐vesicular routes to ferry lipids from their place of synthesis to their destination. For instance, the major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), can be produced by multiple pathways and, in the case of PE, also at multiple locations. However, the molecular components that underlie lipid homeostasis as well as the routes allowing their distribution remain unclear. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. Among the candidates identified, we find genes linked to transcriptional regulation of lipid homeostasis, lipid metabolism, and transport. In particular, we identify a requirement for Csf1—an uncharacterized protein harboring a Chorein‐N lipid transport motif—for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating Csf1 in interorganelle lipid transport and homeostatic adaptation.

Choline Kinetics in Neonatal Liver, Brain and Lung-Lessons from a Rodent Model for Neonatal Care.

Choline requirements are high in the rapidly growing fetus and preterm infant, mainly serving phosphatidylcholine (PC) synthesis for parenchymal growth and one-carbon metabolism via betaine. However, choline metabolism in critical organs during rapid growth is poorly understood. Therefore, we investigated the kinetics of D9-choline and its metabolites in the liver, plasma, brain and lung in 14 d old rats. Animals were intraperitoneally injected with 50 mg/kg D9-choline chloride and sacrificed after 1.5 h, 6 h and 24 h. Liver, plasma, lungs, cerebrum and cerebellum were analyzed for D9-choline metabolites, using tandem mass spectrometry. In target organs, D9-PC and D9-betaine comprised 15.1 ± 1.3% and 9.9 ± 1.2% of applied D9-choline at 1.5 h. D9-PC peaked at 1.5 h in all organs, and decreased from 1.5–6 h in the liver and lung, but not in the brain. Whereas D9-labeled PC precursors were virtually absent beyond 6 h, D9-PC increased in the brain and lung from 6 h to 24 h (9- and 2.5-fold, respectively) at the expense of the liver, suggesting PC uptake from the liver via plasma rather than local synthesis. Kinetics of D9-PC sub-groups suggested preferential hepatic secretion of linoleoyl-PC and acyl remodeling in target organs. D9-betaine showed rapid turnover and served low-level endogenous (D3-)choline synthesis. In conclusion, in neonatal rats, exogenous choline is rapidly metabolized to PC by all organs. The liver supplies the brain and lung directly with PC, followed by organotypic acyl remodeling. A major fraction of choline is converted to betaine, feeding the one-carbon pool and this must be taken into account when calculating choline requirements.

In search of suitable protein targets for anti-malarial and anti-dengue drug discovery

Selecting suitable targets is the critical first step in drug discovery. However, for malaria and dengue this is quite a challenging step due to a large number of available structures for every disease targets, about 139 crystal structures for dengue and 367 structures for malaria. This study employed a combination of druggability filtering and computational analysis through molecular docking and molecular dynamics (MD) simulations in oder to select suitable drug targets for dengue and malaria. Initially, diverse target structures for dengue and malaria in Protein Data Bank were assessed for their druggability and as the result, druggable targets were chosen. There were three potential druggable targets NS5, NS3 and NS2B-NS3 protease for dengue, and several targets involved in the food vacuole, apicoplast, mitochondria, cytosol, phosphatidylcholine synthesis pathways for malaria. Molecular docking of co-crystallised ligands with these targets was carried out and the complexes of targets satisfied with high binding affinies and RMSD values were selected. Subsequently, MD simulations of these complexes were run for 100 ns and different conformational structures of target proteins during the period of time were extracted for ensemble docking for each target. Re-docking results showed a significant difference in using equilibrium structures and initial structures from PDB. Finally, the suitable target structures were selected, including NS5 MTase for dengue and plasmepsin for malaria which can be used for further drug design studies.

Selenium Deficiency Induces Pathological Cardiac Lipid Metabolic Remodeling and Inflammation.

Selenium (Se) disequilibrium is closely involved in many cardiac diseases, although its in vivo mechanism remains uncertain. Therefore, a pig model is created in order to generate a comprehensive picture of cardiac response to dietary Se deficiency. A total of 24 pigs are divided into two equal groups, which were fed a diet with either 0.007mg kg-1 Se or 0.3mg kg-1 Se for 16 weeks. Se deficiency causes cardiac oxidative stress by blocking glutathione and thioredoxin systems and increases thioredoxin domain-containing protein S-nitrosylation. Energy production is disordered, as free fatty acids are overloaded, the tricarboxylic acid cycle is strengthened, and three respiratory chain proteins enhance S-nitrosylation. Excess free fatty acids lead to increased synthesis of diacylglycerol, phosphatidylcholine, and phosphatidylethanolamine, where the latter two are vulnerable to oxidation and causes an increase in malondialdehyde. Moreover, increased palmitic acid enhances de novo ceramide synthesis and accumulation. Additionally, Se deficiency initiates inflammation via cytosolic DNA-sensing pathways, which activates downstream interferon regulatory factor 7 and nuclear factor kappaB. The present study identifies a lipid metabolic vulnerability and inflammation initiation pathway via Se deficiency, which may provide targets for human redox imbalance-induced cardiac disease treatment.

De novo phosphatidylcholine synthesis in the small intestinal epithelium is required for normal dietary lipid handling and maintenance of the mucosal barrier

Cytidine triphosphate:phosphocholine cytidylyltransferase-α (CTα) is the rate limiting enzyme in the major pathway for de novo phosphatidylcholine (PC) synthesis. When CTα is deleted specifically in intestinal epithelial cells of adult mice (CTαIKO mice) fed a high-fat diet they present with weight loss, lipid malabsorption, and high postprandial GLP-1 levels. The current study aimed to characterize the changes that occur in the small intestines of CTαIKO mice using transcriptomics and to determine whether intestinal function could be rescued in CTαIKO mice. We found that impaired de novo PC synthesis in the gut is linked to lower abundance of transcripts related to lipid metabolism and higher abundance of transcripts related to ER stress and cell death, together with loss of goblet cells from the small intestinal epithelium. Furthermore, impaired movement of fatty acids from the intestinal lumen into enterocytes was observed in isolated intestinal sacs derived from CTαIKO mice, a model that excludes factors such as bile, gastric emptying, the nervous system, and circulating hormones. Antibiotic treatment prevented acute weight loss and normalized jejunum TG concentrations after refeeding but did not prevent ER stress or loss of goblet cells in CTαIKO mice. Dietary PC supplementation partially prevented loss of goblet cells but was unable to normalize jejunal TG concentrations after refeeding in CTαIKO mice. High postprandial plasma GLP-1 levels were present in CTαIKO mice regardless of antibiotic treatment, dietary PC content, or dietary fat content. Together, these data show that there is a specific requirement from de novo PC synthesis in maintaining small intestinal homeostasis, including dietary lipid uptake, normal hormone secretion, and barrier function.

PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in Plasmodium falciparum.

PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P.falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.

Characterization of Molecular Properties and Expression of Gene GmPLMT and Its Effects on the Production of Lipid Metabolites in Soybean and Arabidopsis thaliana

Phospholipid N-methyltransferase (PLMT) plays an important role in the synthesis of phosphatidylcholine (PtdCho). The aim of this study was to characterize the molecular properties of GmPLMT and the expression of soybean GmPLMT and its effects on the production of lipid metabolites. Results showed that GmPLMT composed of mainly α-helix was a hydrophobic and transmembrane protein. In soybean leaves, GmPLMT was highly expressed during seedling and flowering stages. In transgenic Arabidopsis thaliana, the highest and lowest expression levels of GmPLMT were detected at flowering and maturity stages, respectively. The total phospholipid contents in soybean grains were decreased from 7.2% (35 days after flowering) to 4.8% (55 days after flowering) and then increased to 7.0% (75 days after flowering). The contents of PtdCho showed a similar pattern to that of total phospholipids. In transgenic A. thaliana seeds, the contents of total phospholipids and PtdCho were significantly increased. Significantly positive correlations were revealed between expression of GmPLMT and contents of both PtdCho and crude fats, and between the contents of PtdCho and both linoleic acid and linolenic acid, suggesting that increased expression of GmPLMT improved the production of lipid metabolites. This study provided solid experimental evidence for further improvement of soybean quality based on GmPLMT in the molecular breeding of soybeans.

Estrogenic mixtures induce alterations in lipidomic profiles in the gonads of female oysters

This study aimed to reveal possible alterations to lipidomic profiles in Sydney rock oysters, Saccostrea glomerata, exposed to estrogenic mixtures (i.e., estrone, E1; 17β-estradiol, E2; estriol, E3; 17α-ethinylestradiol, EE2; bisphenol A, BPA; 4-t-octylphenol, 4-t-OP; and 4-nonylphenol, 4-NP) at “low” and “high” concentrations, typical of those detected in Australian and global receiving waters. A seven-day acute exposure window exhibited significantly lower abundances of many non-polar metabolites in digestive gland, gills, and gonads. Overall, there was a strong effect of the carrier solvent ethanol (despite a low exposure of 0.0002%), with all solvent containing treatments exhibiting lower abundances of lipidic metabolites, especially in the gill and digestive gland. No significant changes of the lipidome were exhibited in the male gonad by estrogenic exposure. However, in the female gonad, significant reductions of phospholipids and phosphatidylcholine were associated with exposure to high estrogenic mixtures. We hypothesise that the decreases in these phospholipids in the female gonad may be attributable to 1) lower algal consumption and thus lower uptake of lipidic building blocks; 2) a reduction of available substrates for phospholipid and phosphatidylcholine synthesis; and/or 3) induction of reactive oxygen species via estrogen metabolism, which may cause lipid peroxidation and lower abundance of phospholipids.

GlmS mediated knock-down of a phospholipase expedite alternate pathway to generate phosphocholine required for phosphatidylcholine synthesis in Plasmodium falciparum.

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from the catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase (PfLPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP- targeting approach, PfLPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of PfLPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that PfLPL20 is not essential for the parasite. However, in PfLPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with up-regulation of phosphocholine and SAM levels; these results show up-regulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights the presence of alternate pathways for lipid homeostasis/membrane-biogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite.

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline‐auxotroph yeast

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl‐CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl‐CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine‐tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity.

The Phospholipid N-Methyltransferase and Phosphatidylcholine Synthase Pathways and the ChoXWV Choline Uptake System Involved in Phosphatidylcholine Synthesis Are Widely Conserved in Most, but Not All Brucella Species.

The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts.

Differential metabolism of choline supplements in adult volunteers

BackgroundAdequate intake of choline is essential for growth and homeostasis, but its supply does often not meet requirements. Choline deficiency decreases phosphatidylcholine (PC) and betaine synthesis, resulting in organ pathology, especially of liver, lung, and brain. This is of particular clinical importance in preterm infants and cystic fibrosis patients. We compared four different choline supplements for their impact on plasma concentration and kinetics of choline, betaine as a methyl donor and trimethylamine oxide (TMAO) as a marker of bacterial degradation prior to absorption.MethodsProspective randomized cross-over study (1/2020–4/2020) in six healthy adult men. Participants received a single dose of 550 mg/d choline equivalent in the form of choline chloride, choline bitartrate, α-glycerophosphocholine (GPC), and egg-PC in randomized sequence at least 1 week apart. Blood was taken from t = − 0.1–6 h after supplement intake. Choline, betaine, TMAO, and total PC concentrations were analyzed by tandem mass spectrometry. Results are shown as medians and interquartile range.ResultsThere was no difference in the AUC of choline plasma concentrations after intake of the different supplements. Individual plasma kinetics of choline and betaine differed and concentrations peaked latest for PC (at ≈3 h). All supplements similarly increased plasma betaine. All water-soluble supplements rapidly increased TMAO, whereas egg-PC did not.ConclusionAll supplements tested rapidly increased choline and betaine levels to a similar extent, with egg-PC showing the latest peak. Assuming that TMAO may have undesirable effects, egg-PC might be best suited for choline supplementation in adults.Study registrationThis study was registered at “Deutsches Register Klinischer Studien” (DRKS) (German Register for Clinical Studies), 17.01.2020, DRKS00020454.

Triacylglycerol remodeling in Physaria fendleri indicates oil accumulation is dynamic and not a metabolic endpoint.

Oilseed plants accumulate triacylglycerol (TAG) up to 80% of seed weight with the TAG fatty acid composition determining its nutritional value or use in the biofuel or chemical industries. Two major pathways for production of diacylglycerol (DAG), the immediate precursor to TAG, have been identified in plants: de novo DAG synthesis and conversion of the membrane lipid phosphatidylcholine (PC) to DAG, with each pathway producing distinct TAG compositions. However, neither pathway fits with previous biochemical and transcriptomic results from developing Physaria fendleri seeds for accumulation of TAG containing >60% lesquerolic acid (an unusual 20 carbon hydroxylated fatty acid), which accumulates at only the sn-1 and sn-3 positions of TAG. Isotopic tracing of developing P. fendleri seed lipid metabolism identified that PC-derived DAG is utilized to initially produce TAG with only one lesquerolic acid. Subsequently a nonhydroxylated fatty acid is removed from TAG (transiently reproducing DAG) and a second lesquerolic acid is incorporated. Thus, a dynamic TAG remodeling process involving anabolic and catabolic reactions controls the final TAG fatty acid composition. Reinterpretation of P. fendleri transcriptomic data identified potential genes involved in TAG remodeling that could provide a new approach for oilseed engineering by altering oil fatty acid composition after initial TAG synthesis; and the comparison of current results to that of related Brassicaceae species in the literature suggests the possibility of TAG remodeling involved in incorporation of very long-chain fatty acids into the TAG sn-1 position in various plants.