Species Of Phosphatidylglycerol Research Articles

Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids.

We show that Staphylococcus aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerols bearing one or two degrees of unsaturation derived from the incorporation of C18:1Δ9 at 12°C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance the growth of S. aureus in the food environment.

Acute myocardial infarction preferentially alters low-abundant, long-chain unsaturated phospholipid and sphingolipid species in plasma high-density lipoprotein subpopulations

AimHigh-density lipoprotein (HDL) particles in ST-segment elevation myocardial infarction (STEMI) are deficient in their anti-atherogenic function. Molecular determinants of such deficiency remain obscure. MethodsFive major HDL subpopulations were isolated using density-gradient ultracentrifugation from STEMI patients (n = 12) and healthy age- and sex-matched controls (n = 12), and 160 species of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, sphingomyelin and ceramide were quantified by LC-MS/MS. ResultsMultiple minor species of proinflammatory phosphatidic acid and lysophosphatidylcholine were enriched by 1.7–27.2-fold throughout the majority of HDL subpopulations in STEMI. In contrast, minor phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin and ceramide species were typically depleted up to 3-fold in STEMI vs. control HDLs, while abundances of their major species did not differ between the groups. Intermediate-to-long-chain phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol species were more affected by STEMI than their short-chain counterparts, resulting in positive correlations between their fold decrease and the carbon chain length. Additionally, fold decreases in the abundances of multiple lipid species were positively correlated with the double bond number in their carbon chains. Finally, abundances of several phospholipid and ceramide species were positively correlated with cholesterol efflux capacity and antioxidative activity of HDL subpopulations, both reduced in STEMI vs controls. KEGG pathway analysis tied these species to altered glycerophospholipid and linoleic acid metabolism. ConclusionsMinor unsaturated intermediate-to-long-chain phospholipid and sphingolipid species in HDL subpopulations are most affected by STEMI, reflecting alterations in glycerophospholipid and linoleic acid metabolism with the accumulation of proinflammatory lysolipids and maintenance of homeostasis of major phospholipid species.

Coral Lipidome: Molecular Species of Phospholipids, Glycolipids, Betaine Lipids, and Sphingophosphonolipids.

Coral reefs are the most biodiversity-rich ecosystems in the world's oceans. Coral establishes complex interactions with various microorganisms that constitute an important part of the coral holobiont. The best-known coral endosymbionts are Symbiodiniaceae dinoflagellates. Each member of the coral microbiome contributes to its total lipidome, which integrates many molecular species. The present study summarizes available information on the molecular species of the plasma membrane lipids of the coral host and its dinoflagellates (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), ceramideaminoethylphosphonate, and diacylglyceryl-3-O-carboxyhydroxymethylcholine), and the thylakoid membrane lipids of dinoflagellates (phosphatidylglycerol (PG) and glycolipids). Alkyl chains of PC and PE molecular species differ between tropical and cold-water coral species, and features of their acyl chains depend on the coral's taxonomic position. PS and PI structural features are associated with the presence of an exoskeleton in the corals. The dinoflagellate thermosensitivity affects the profiles of PG and glycolipid molecular species, which can be modified by the coral host. Coral microbiome members, such as bacteria and fungi, can also be the source of the alkyl and acyl chains of coral membrane lipids. The lipidomics approach, providing broader and more detailed information about coral lipid composition, opens up new opportunities in the study of biochemistry and ecology of corals.

The anti-inflammatory and antiviral properties of anionic pulmonary surfactant phospholipids.

The pulmonary surfactant system of the lung is a lipid and protein complex, which regulates the biophysical properties of the alveoli to prevent lung collapse and the innate immune system in the lung. Pulmonary surfactant is a lipoprotein complex consisting of 90% phospholipids and 10% protein, by weight. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), exist at very high concentrations in the extracellular alveolar compartments. We have reported that one of the most dominant molecular species of PG, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and PI inhibit inflammatory responses induced by multiple toll-like receptors (TLR2/1, TLR3, TLR4, and TLR2/6) by interacting with subsets of multiprotein receptor components. These lipids also exert potent antiviral effects against RSV and influenza A, in vitro, by inhibiting virus binding to host cells. POPG and PI inhibit these viral infections in vivo, in multiple animal models. Especially noteworthy, these lipids markedly attenuate SARS-CoV-2 infection including its variants. These lipids are natural compounds that already exist in the lung and, thus, are less likely to cause adverse immune responses by hosts. Collectively, these data demonstrate that POPG and PI have strong potential as novel therapeutics for applications as anti-inflammatory compounds and preventatives, as treatments for broad ranges of RNA respiratory viruses.

Modulations of bioactive lipids and their receptors in postmortem Alzheimer's disease brains.

Analyses of brain samples from Alzheimer's disease (AD) patients may be expected to help us improve our understanding of the pathogenesis of AD. Bioactive lipids, including sphingolipids, glycerophospholipids, and eicosanoids/related mediators have been demonstrated to exert potent physiological actions and to be involved in the pathogenesis of various human diseases. In this cross-sectional study, we attempted to elucidate the associations of these bioactive lipids with the pathogenesis/pathology of AD through postmortem studies of human brains. We measured the levels of glycerophospholipids, sphingolipids, and eicosanoids/related mediators in the brains of patients with AD (AD brains), patients with Cerad score B (Cerad-b brains), and control subjects (control brains), using a liquid chromatography-mass spectrometry method; we also measured the mRNA levels of specific receptors for these bioactive lipids in the same brain specimens. The levels of several species of sphingomyelins and ceramides were higher in the Cerad-b and AD brains. Levels of several species of lysophosphatidic acids (LPAs), lysophosphatidylcholine, lysophosphatidylserine, lysophosphatidylethanolamine (LPE), lysophosphatidylinositol, phosphatidylcholine, phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylglycerol were especially high in the Cerad-b brains, while those of lysophosphatidylglycerol (LPG) were especially high in the AD brains. Several eicosanoids, including metabolites of prostaglandin E2, oxylipins, metabolites of epoxide, and metabolites of DHA and EPA, such as resolvins, were also modulated in the AD brains. Among the lipid mediators, the levels of S1P2, S1P5, LPA1, LPA2, LPA6, P2Y10, GPR174, EP1, DP1, DP2, IP, FP, and TXA2r were lower in the AD and/or Cerad-b brains. The brain levels of ceramides, LPC, LPI, PE, and PS showed strong positive correlations with the Aβ contents, while those of LPG showed rather strong positive correlations with the presence of senile plaques and neurofibrillary tangles. A discriminant analysis revealed that LPG is especially important for AD and the LPE/PE axis is important for Cerad-b. Comprehensive lipidomics, together with the measurement of lipid receptor expression levels provided novel evidence for the associations of bioactive lipids with AD, which is expected to facilitate future translational research and reverse translational research.

Light modulates the lipidome of the photosynthetic sea slug Elysia timida

Long-term kleptoplasty, the capability to retain functional stolen chloroplasts (kleptoplasts) for several weeks to months, has been shown in a handful of Sacoglossa sea slugs. One of these sea slugs is Elysia timida, endemic to the Mediterranean, which retains functional chloroplasts of the macroalga Acetabularia acetabulum. To understand how light modulates the lipidome of E. timida, sea slug specimens were subjected to two different 4-week light treatments: regular light and quasi-dark conditions. Lipidomic analyses were performed by HILIC-HR-ESI-MS and MS/MS. Quasi-dark conditions caused a reduction in the amount of essential lipids for photosynthetic membranes, such as glycolipids, indicating high level of kleptoplast degradation under sub-optimal light conditions. However, maximum photosynthetic capacities (Fv/Fm) were identical in both light treatments (≈0.75), showing similar kleptoplast functionality and suggesting that older kleptoplasts were targeted for degradation. Although more stable, the phospholipidome showed differences between light treatments: the amount of certain lipid species of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylglycerol (PG) decreased under quasi-dark conditions, while other lipid species of phosphatidylcholine (PC), PE and lyso-PE (LPE) increased. Quasi-dark conditions promoted a decrease in the relative abundance of polyunsaturated fatty acids. These results suggest a light-driven remodelling of the lipidome according to the functions of the different lipids and highlight the plasticity of polar lipids in the photosynthetic sea slug E. timida.

Lipidomic analysis of surfactant and plasma from horses with asthma and age-matched healthy horses.

To perform lipidomic analysis of surfactant and plasma from asthmatic and healthy horses. 30 horses with clinical signs of asthma and 30 age-matched control horses. Detailed history, physical examination, CBC, and bronchoalveolar lavage fluid (BALF) cytologies were obtained. Asthmatic horses were grouped based on their BALF inflammatory profile: severe equine asthma (SEA), mild equine asthma with neutrophilic airway inflammation (MEA-N), or mild equine asthma with eosinophilic airway inflammation (MEA-E). Each asthma group was assigned its own age-matched control group. Lipidomic analysis was completed on surfactant and plasma. Surfactant protein D (SP-D) concentrations were measured in serum and BALF. SEA surfactant was characterized by a phospholipid deficit and altered composition (increased ceramides, decreased phosphatidylglycerol, and increased cyclic phosphatidic acid [cPA]). In comparison, MEA-N surfactant only had a decrease in select phosphatidylglycerol species and increased cPA levels. The plasma lipidomic profile was significantly different in all asthma groups compared to controls. Specifically, all groups had increased plasma phytoceramide. SEA horses had increased plasma cPA and diacylglycerol whereas MEA-N horses only had increased cPA. MEA-E horses had increases in select ceramides and dihydrocermides. Only SEA horses had significantly increased serum SP-D concentrations. The most significant surfactant alterations were present in SEA (altered phospholipid content and composition); only mild changes were observed in MEA-N horses. The plasma lipidomic profile was significantly altered in all groups of asthmatic horses and differed among groups. Data from a larger population of asthmatic horses are needed to assess implications for diagnosis, prognosis, and treatment.

Listeria monocytogenes exposed to antimicrobial peptides displays differential regulation of lipids and proteins associated to stress response.

With the onset of Listeria monocytogenes resistance to the bacteriocin nisin, the search for alternative antimicrobial treatments is of fundamental importance. In this work, we set out to investigate proteins and lipids involved in the resistance mechanisms of L. monocytogenes against the antimicrobial peptides (AMPs) nisin and fengycin. The effect of sub-lethal concentrations of nisin and lipopeptide fengycin secreted by Bacillus velezensis P34 on L. monocytogenes was investigated by mass spectrometry-based lipidomics and proteomics. Both AMPs caused a differential regulation of biofilm formation, confirming the promotion of cell attachment and biofilm assembling after treatment with nisin, whereas growth inhibition was observed after fengycin treatment. Anteiso branched-chain fatty acids were detected in higher amounts in fengycin-treated samples (46.6%) as compared to nisin-treated and control samples (39.4% and 43.4%, respectively). In addition, a higher relative abundance of 30:0, 31:0 and 32:0 phosphatidylglycerol species was detected in fengycin-treated samples. The lipidomics data suggest the inhibition of biofilm formation by the fengycin treatment, while the proteomics data revealed downregulation of important cell wall proteins involved in the building of biofilms, such as the lipoteichoic acid backbone synthesis (Lmo0927) and the flagella-related (Lmo0718) proteins among others. Together, these results provide new insights into the modification of lipid and protein profiles and biofilm formation in L. monocytogenes upon exposure to antimicrobial peptides.

Phospholipid Profiles for Phenotypic Characterization of Adipose-Derived Multipotent Mesenchymal Stromal Cells.

Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.

Analysis of verteporfin liposomal formulations for phospholipids and phospholipid degradation products by liquid chromatography-mass spectrometry (LC-MS)

Lipid composition and lipid degradation are critical to the stability of liposomal formulations which can impact the safety and efficacy of the drug. Herein we developed and validated an ultrahigh performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) method for determining phospholipid composition and phospholipid degradation products in a verteporfin liposomal formulation (Visudyne). The high mass accuracy (<5 ppm) of the QTOF method coupled with database searching (SimLipid) and comparison with known standards accurately identified and quantified the phospholipid compositions and lipid degradation products. The analysis of Visudyne indicated that more than 50% (w/w) of the total phospholipids are composed of phosphatidylcholine (PC) 14:0–14:0 and major phosphatidylglycerol (PG) species found are PG 16:0–18:2, PG 16:0–18:1, PG 18:0–18:2, and PG 18:0–18:1. The LC-MS method developed is capable of separating structural isomers such as PG 18:1–18:1 versus PG 18:0–18:2 and the separation of PG stereoisomers, such as PG 18:1–18:1 cis and PG 18:1–18:1 trans. The major lipid degradation products in Visudyne includes lysophosphatidylcholine and a few saturated and unsaturated lysophosphatidylglycerols, and free fatty acids (FFA). Each degradation product is less than 1% of the total phospholipids (w/w). In addition, the lipid profiles of naturally sourced egg PG from six different vendors were compared with the PG composition in Visudyne. Differences in lipid composition in egg PGs from different vendors were observed and the PG composition in Visudyne is matched with the lipid profile of the some of the egg PGs from different vendors. Drug developers can utilize this method to assess raw materials and lipid-based drug product quality and regulatory scientists can monitor the quality of the drug available in the market using this validated method.

Abstract 2342: Multidimensional metabolomic stratification of ER+/HER2- compared to ER-/HER2- breast tumors

Abstract Introduction: In the United States, breast cancer represents the leading cancer diagnosis among women and can readily be classified as a metabolic disease based on its distinct metabolic activity within the tumor microenvironment. Compared to other omics technologies, extensive lipidomic and metabolomic studies are lacking. Here in, we evaluated a cohort of 109 tumors characterized as ER+/HER2- and ER-/HER2- based on immunohistochemistry (IHC) and performed comprehensive structural lipidomic, signaling lipidomic, and global metabolomic analyses for an extensive characterization of the biophysical, signaling, and metabolic interplay between these tumors. Methods: Clinical IHC subtyping of core biopsies was used to select a cohort of patients with ER+/HER2- or ER-/HER2- primary tumors from flash-frozen surgical samples. The positive/negative status of ER/PR/HER2 was defined using updated ASCO 2020 guidelines. Ki-67 status was determined using the 2011 St. Gallen's International Expert Consensus recommendations. ER low (1-10%) cases were excluded from this analysis. Structural lipidomic analysis was employed through the use of MS/MSALL high resolution shotgun lipidomics using a SCIEX 5600+ TripleTOF micro LC approach characterizing 23 lipid classes and over 1200 molecular species. Signaling lipids were analyzed using a SCIEX 6600 TripleTOF microLC platform characterizing 106 lipid analytes across octadecanoid, eicosanoid and docosanoid species. Metabolomics analysis was performed using LECO PEGASUS GC TOF, SCIEX 5500 HILIC LC MS/MS analysis, and SCIEX 6600 High resolution RP-LC-MS analysis detecting 450 metabolite Metabolomics data was further interpreted using MetaboAnalyst software. Results/Conclusions: Compared to their ER+ counterparts, ER-/HER2- tumors exhibited a significant decrease in triacylglycerides, and a corresponding increase in cholesterol ester, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol species demonstrating a signature of biophysical and metabolic rewiring with alterations in Kennedy pathway lipid shuttling. One signaling lipid was decreased and six were increased (predominantly arachidonic species) in ER-/HER2- tumors compared to ER+/HER2- ones. Metabolomic analysis revealed distinct alterations in cysteine/methionine, arginine/proline, purine, butanoate, and tryptophan metabolism. Utilizing a multidimensional metabolic integration approach, we identified distinct biophysical, signaling, and biochemical alterations in ER+/HER2- compared to ER-/HER2- breast tumors, which may impact selection of therapy and outcome in the future. Citation Format: Valerie Bussberg, Vladimir Tolstikov, Guisong Wang, Punit Shah, Rick Searfoss, Leigh Fantacone-Campbell, Jeffrey A. Hooke, Brenda Deyarmin, Rebecca N. Zingmark, Stella Somiari, Jianfang Liu, Leonid Kvecher, Bradley Mostoller, Lori Sturtz, Praveen-Kumar Raj-Kumar, Elder Granger, Linda Vahdat, Mary L. Cutler, Chas Bountra, Rangaprasad Sarangarajan, Hai Hu, Albert J. Kovatich, Michael A. Kiebish, Niven R. Narain, Craig D. Shriver. Multidimensional metabolomic stratification of ER+/HER2- compared to ER-/HER2- breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2342.

Revealing Fatty Acid Heterogeneity in Staphylococcal Lipids with Isotope Labeling and RPLC-IM-MS.

Up to 80% of the fatty acids in Staphylococcus aureus membrane lipids are branched, rather than straight-chain, fatty acids. The branched fatty acids (BCFAs) may have either an even or odd number of carbons, and the branch position may be at the penultimate carbon (iso) or the antepenultimate (anteiso) carbon of the tail. This results in two sets of isomeric fatty acid species with the same number of carbons that cannot be resolved by mass spectrometry. The isomer/isobar challenge is further complicated when the mixture of BCFAs and straight-chain fatty acids (SCFAs) are esterified into diacylated lipids such as the phosphatidylglycerol (PG) species of the S. aureus membrane. No conventional chromatographic method has been able to resolve diacylated lipids containing mixtures of SCFAs, anteiso-odd, iso-odd, and iso-even BCFAs. A major hurdle to method development in this area is the lack of relevant analytical standards for lipids containing BCFA isomers. The diversity of the S. aureus lipidome and its naturally high levels of BCFAs present an opportunity to explore the potential of resolving diacylated lipids containing BCFAs and SFCAs. Using our knowledge of lipid and fatty acid biosynthesis in S. aureus, we have used a stable-isotope-labeling strategy to develop and validate a 30 min C18 reversed-phase liquid chromatography method combined with traveling-wave ion mobility-mass spectrometry to provide resolution of diacylated lipids based on the number of BCFAs that they contain.

Colistin Treatment Affects Lipid Composition of Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii (A. baumannii) causes severe and often fatal healthcare-associated infections due partly to antibiotic resistance. There are no studies on A. baumannii lipidomics of susceptible and resistant strains grown at lethal and sublethal concentrations. Therefore, we analyzed the impact of colistin resistance on glycerolipids’ content by using untargeted lipidomics on clinical isolate. Nine lipid sub-classes were annotated, including phosphatidylcholine, rarely detected in the bacterial membrane among 130 different lipid species. The other lipid sub-classes detected are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), lysophosphatidylethanolamine, hemibismonoacylglycerophosphate, cardiolipin, monolysocardiolipin, diacylglycerol, and triacylglycerol. Under lethal and sublethal concentrations of colistin, significant reduction of PE was observed on the resistant and susceptible strain, respectively. Palmitic acid percentage was higher at colistin at low concentration but only for the susceptible strain. When looking at individual lipid species, the most abundant PE and PG species (PE 34:1 and PG 34:1) are significantly upregulated when the susceptible and the resistant strains are cultivated with colistin. This is, to date, the most exhaustive lipidomics data compilation of A. baumannii cultivated in the presence of colistin. This work is highlighting the plasma membrane plasticity used by this gram-negative bacterium to survive colistin treatment.

Study of phospholipid molecular species of the green seaweed \(\textit{Halimeda incrassata}\) Lamx. from Truong Sa islands, Vietnam

In this report, by a high performance liquid chromatography (HPLC) - high resolution mass spectrometry (HRMS) method, 7 molecular species of phospholipid in the polar lipid class were identified including phosphatidylinositol (PI) and phosphatidylglycerol (PG). The PI 32:0 (16:0/16:0) and PG 34:3 (16:1/18:2) molecular species have the highest content. PI molecular species are mainly formed by saturated fatty acids (16:0, 18:0) and only one polyunsaturated fatty acids C20:4, while PG species are formed by unsaturated fatty acids (16:1, 18:2 and 18:3) and only one saturated fatty acid C16:0.

Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.

Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.

Phosphatidylinositol synthesis, its selective salvage, and inter-regulation of anionic phospholipids in Toxoplasma gondii

Phosphatidylinositol (PtdIns) serves as an integral component of eukaryotic membranes; however, its biosynthesis in apicomplexan parasites remains poorly understood. Here we show that Toxoplasma gondii—a common intracellular pathogen of humans and animals—can import and co-utilize myo-inositol with the endogenous CDP-diacylglycerol to synthesize PtdIns. Equally, the parasite harbors a functional PtdIns synthase (PIS) containing a catalytically-vital CDP-diacylglycerol phosphotransferase motif in the Golgi apparatus. Auxin-induced depletion of PIS abrogated the lytic cycle of T. gondii in human cells due to defects in cell division, gliding motility, invasion, and egress. Isotope labeling of the PIS mutant in conjunction with lipidomics demonstrated de novo synthesis of specific PtdIns species, while revealing the salvage of other lipid species from the host cell. Not least, the mutant showed decline in phosphatidylthreonine, and elevation of selected phosphatidylserine and phosphatidylglycerol species, indicating a rerouting of CDP-diacylglycerol and homeostatic inter-regulation of anionic phospholipids upon knockdown of PIS. In conclusion, strategic allocation of own and host-derived PtdIns species to gratify its metabolic demand features as a notable adaptive trait of T. gondii. Conceivably, the dependence of T. gondii on de novo lipid synthesis and scavenging can be exploited to develop new anti-infectives.

Separation and identification of diacylglycerols containing branched chain fatty acids by liquid chromatography-mass spectrometry

A combination of two chromatographic and two enzymatic methods was used for the analysis of molecular species of lipids from Gram-positive bacteria of the genus Kocuria. Gram-positive bacteria contain a majority of branched fatty acids (FAs), especially iso- and/or anteiso-FAs. Two strains K. rhizophila were cultivated at three different temperatures (20, 28, and 37°C) and the majority phospholipid, i.e., the mixture of molecular species of phosphatidylglycerols (PGs) was separated by means of hydrophilic interaction liquid chromatography (HILIC). After enzymatic hydrolysis of PGs by phospholipase C and derivatization of the free OH group, the sn-1,2-diacyl-3-acetyl triacylglycerols (AcTAGs) were separated by reversed phase HPLC. Molecular species such as i-15:0/i-15:0/2:0, ai-15:0/ai-15:0/2:0, and 15:0/15:0/2:0 (straight chains) were identified by liquid chromatography-positive electrospray ionization mass spectrometry. The tandem mass spectra of both standards and natural compounds containing iso, anteiso and straight chain FAs with the same carbons were identical. Therefore, for identification of the ratio of two regioisomers, i.e. i-15:0/ai-15:0/2:0 vs. ai-15:0/i-15:0/2:0, they were cleavage by pancreatic lipase. The mixture of free fatty acids (FFAs) and 2-monoacylglycerols (2-MAGs) was obtained. After their separation by TLC and esterification and/or transesterification, the fatty acid methyl esters were quantified by GC-MS and thus the ratio of regioisomers was determined.It has been shown that the ratio of PG (containing as majority i-15: 0 / i-15: 0, i-15: 0 / ai-15: 0 and / or ai-15: 0 / i-15: 0 and ai-15: 0 / ai-15: 0 molecular species) significantly affected the membrane flow of bacterial cells cultured at different temperatures.

Photosynthetic pigment production and metabolic and lipidomic alterations in the marine cyanobacteria Synechocystis sp. PCC 7338 under various salinity conditions

Synechocystis sp. PCC 7338 (hereafter referred to as Synechocystis 7338) is a marine cyanobacterium that has the potential to produce photosynthetic pigments. In this study, we investigated the effects of various NaCl concentrations (0, 0.4, 0.8, and 1.2 M) on cell growth, photosynthetic pigments, and metabolites and intact lipid species profiles in Synechocystis 7338. The overall growth pattern of Synechocystis 7338 was similar under 0, 0.4, and 0.8 M NaCl conditions. Cell growth was retarded after reaching the exponential phase under 1.2 M NaCl; however, a similar growth pattern was observed after the exponential phase under 0.4 M NaCl (control group). The highest production of chlorophyll a (4.18 mg L−1), allophycocyanin (4.08 mg L−1), and phycoerythrin (1.70 mg L−1) were achieved under 1.2 M NaCl conditions. Altered metabolic and lipidomic profiles were observed at different NaCl conditions; significantly increased relative yields of glucosylglycerol, one diacylglyceryltrimethylhomoserine, one monogalactosyldiacylglycerol, and four phosphatidylglycerol species were observed under 1.2 M NaCl conditions using gas chromatography–mass spectrometry and direct infusion–mass spectrometry analyses. In addition, it was revealed that the photosynthetic activity recovered under 1.2 M NaCl conditions in long-term culture. Hydrogen peroxide content significantly increased under 1.2 M NaCl conditions. It is believed that glutathione content also significantly increased under high salinity conditions to retain the normal functioning of Synechocystis 7338. These results indicate that high salinity conditions for Synechocystis 7338 culture could be used for the large-scale production of chlorophyll a, allophycocyanin, phycoerythrin, and other bioactive metabolites.

Decreased R:FR Ratio in Incident White Light Affects the Composition of Barley Leaf Lipidome and Freezing Tolerance in a Temperature-Dependent Manner

In cereals, C-repeat binding factor genes have been defined as key components of the light quality-dependent regulation of frost tolerance by integrating phytochrome-mediated light and temperature signals. This study elucidates the differences in the lipid composition of barley leaves illuminated with white light or white light supplemented with far-red light at 5 or 15 °C. According to LC-MS analysis, far-red light supplementation increased the amount of monogalactosyldiacylglycerol species 36:6, 36:5, and 36:4 after 1 day at 5 °C, and 10 days at 15 °C resulted in a perturbed content of 38:6 species. Changes were observed in the levels of phosphatidylethanolamine, and phosphatidylserine under white light supplemented with far-red light illumination at 15 °C, whereas robust changes were observed in the amount of several phosphatidylserine species at 5 °C. At 15 °C, the amount of some phosphatidylglycerol species increased as a result of white light supplemented with far-red light illumination after 1 day. The ceramide (42:2)-3 content increased regardless of the temperature. The double-bond index of phosphatidylglycerol, phosphatidylserine, phosphatidylcholine ceramide together with total double-bond index changed when the plant was grown at 15 °C as a function of white light supplemented with far-red light. white light supplemented with far-red light increased the monogalactosyldiacylglycerol/diacylglycerol ratio as well. The gene expression changes are well correlated with the alterations in the lipidome.

Lipidome-wide characterization of phosphatidylinositols and phosphatidylglycerols on C[dbnd]C location level

Phosphatidylglycerol (PG) and phosphatidylinositol (PI) are two essential classes of glycerophospholipids (GPs), playing versatile roles such as signalling messengers and lipid-protein interaction ligands in cell. Although a majority of PG and PI molecular species contain unsaturated fatty acyl chain(s), conventional tandem mass spectrometry (MS/MS) methods cannot discern isomers different in carbon-carbon double bond (CC) locations. In this work, we paired phosphate methylation with acetone Paternò–Büchi (PB) reaction, aiming to provide a solution for sensitive and structurally informative analysis of these two important classes of GPs down to the location of CC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow was established. Offline methylated PG or PI mixtures were subjected to hydrophilic interaction chromatographic separation, online acetone PB reaction, and MS/MS via collision-induced dissociation (CID) for CC location determination in positive ion mode. This method was sensitive, offering limit of identification at 5 nM for both PG and PI standards down to CC locations. On molecular species level, 49 PI and 31 PG were identified from bovine liver, while 61 PIs were identified from human plasma. This workflow also enabled ratiometric comparisons of CC location isomers (C18:1 Δ9 vs. Δ11) of a series of PIs from type 2 diabetes (T2D) plasma to that of normal plasma samples. PI 16:0_18:1 and PI 18:0_18:1 were found to exhibit significant changes in CC isomeric ratios between T2D and normal plasma samples. The above results demonstrate that the developed LC-PB-MS/MS workflow is applicable to different classes of lipids and compatible with other established lipid derivatization methods to achieve comprehensive lipid analysis.