HIV-1 Gag MA domain binds to cardiolipin in a binding mode distinct from virus assemble mediator PI(4,5)P2.

The human immunodeficiency virus type 1 (HIV-1) Gag protein is responsible for facilitating HIV-1 virion assembly and budding. Our study demonstrates that cardiolipin (CL), a component found in the inner mitochondrial membrane, exhibits the highest binding affinity to the N-terminal MA domain of the HIV-1 Gag protein within the lipid group of host cells. To assess this binding interaction, we synthesized short acyl chain derivatives of CL and employed surface plasmon resonance (SPR) analysis to determine the dissociation constants (Kd) for CL and the MA domain. Simultaneously, we examined the Kd of D-myo-phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) derivatives, known to play a crucial role in virion formation. Among all the derivatives, Tetra-C7 -CL exhibited the lowest Kd value (Kd = 30.8 ± 6.9 µM) for MA binding on the CL analog-immobilized sensorchip, indicating a higher affinity. Similarly, the Kd value of Di-C7 -PIP2 (Kd = 36.6 ± 4.7 µM) was the lowest on the PI(4,5)P2 analog-immobilized sensorchip. Thus, Tetra-C7 -CL binds to the MA domain using a distinct binding mode while displaying a comparable binding affinity to Di-C7 -PIP2. This discovery holds significant implications for comprehending the virological importance of CL-MA domain binding, such as its subcellular distribution, including mitochondrial translocation, and involvement in viral particle formation in concert with PI(4,5)P2 . Furthermore, this study has the potential to contribute to the development of drugs in the future.

Protein Dynamics Mediated by Cardiolipin in Bacteria.

Bacterial proteins targeting the appropriate subcellular sites are the base for their proper function. Several studies have shown that the anionic phospholipid cardiolipin (CL), a conical lipid preferring negative membrane curvature, modulates the lipid bilayers' structure, which impacts the activity of their resident proteins. Due to the favor of negative membrane curvature, CL is not randomly distributed in the bacterial plasma membrane. In contrast, it gathers in particular parts of the cell membrane to form microdomains, in which many functional membrane proteins are accumulated and carry out diverse physiological processes of bacteria, such as cell division, metabolism, infection, and antibiotic residence. In addition, CL has a unique structure that carries two negative charges, which makes it play a pivotal role in protein assembly, interaction, and location. These characteristics of CL make it closely related to many crucial physiological functions of bacteria. Here, we have reviewed the mechanism of protein dynamics mediated by CL initiated on the bacterial membrane. Furthermore, we studied the effect of CL on bacterial infection and antibiotic residence. Finally, the CL-targeting therapeutic agents for antibacterial therapy are also examined.

Visualization of Differential Cardiolipin Profiles in Murine Retinal Cell Layers by High-Resolution MALDI Mass Spectrometry Imaging.

The precise fatty acyl chain configuration of cardiolipin (CL), a tetrameric mitochondrial-specific membrane lipid, exhibits dependence on cell and tissue types. A powerful method to map CL profiles in tissue sections in a spatially resolved manner is matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). To build on and explore this potential, we employed a quadrupole time-of-flight mass spectrometer along with optimized sample preparation protocols. We imaged the CL profiles of individual murine retinal cell layers at a pixel size of 10 µm. In combination with tandem MS, we obtained detailed insights into the CL composition of individual retinal cell layers. In particular, we found differential expression of the polyunsaturated fatty acids (PUFA) linoleic, arachidonic, and docosahexaenoic acids. PUFAs are prone to peroxidation and hence regarded as critical factors in development and progression of retinal pathologies, such as age-related macular degeneration (AMD). The ability of MALDI-MSI to provide cues on the CL composition in neuronal tissue with close to single-cell resolution can provide important insights into retinal physiology in health and disease.

Endoplasmic reticulum stress and mitochondrial dysfunction during aging: Role of sphingolipids.

The endoplasmic reticulum (ER) plays a key role in the regulation of protein folding, lipid synthesis, calcium homeostasis, and serves as a primary site of sphingolipid biosynthesis. ER stress (ER dysfunction) participates in the development of mitochondrial dysfunction during aging. Mitochondria are in close contact with the ER through shared mitochondria associated membranes (MAM). Alteration of sphingolipids contributes to mitochondria-driven cell injury. Cardiolipin is a phospholipid that is critical to maintain enzyme activity in the electron transport chain. The aim of the current study was to characterize the changes in sphingolipids and cardiolipin in ER, MAM, and mitochondria during the progression of aging in young (3 mo.), middle (18 mo.), and aged (24 mo.) C57Bl/6 mouse hearts. ER stress increased in hearts from 18 mo. mice and mice exhibited mitochondrial dysfunction by 24 mo. Hearts were pooled to isolate ER, MAM, and subsarcolemmal mitochondria (SSM). LC-MS/MS quantification of lipid content showed that aging increased ceramide content in ER and MAM. In addition, the contents of sphingomyelin and monohexosylceramides are also increased in the ER from aged mice. Aging increased the total cardiolipin content in the ER. Aging did not alter the total cardiolipin content in mitochondria or MAM yet altered the composition of cardiolipin with aging in line with increased oxidative stress compared to young mice. These results indicate that alteration of sphingolipids can contribute to the ER stress and mitochondrial dysfunction that occurs during aging.

Diet Restriction Impact on High-Fat-Diet-Induced Obesity by Regulating Mitochondrial Cardiolipin Biosynthesis and Remodeling.

Diet restriction (DR) ameliorates obesity by regulating mitochondrial function. Cardiolipin (CL), a mitochondrial phospholipid, is closely associated with mitochondrial function. This study aimed to evaluate the anti-obesity effects of graded levels of DR based on mitochondrial CL levels in the liver. Obese mice were treated with 0%, 20%, 40%, and 60% reductions in the normal diet compared to normal animals (0 DR, 20 DR, 40 DR, and 60 DR groups, respectively). Biochemical and histopathological analyses were performed to evaluate the ameliorative effects of DR on obese mice. The altered profile of mitochondrial CL in the liver was explored using a targeted metabolomics strategy by ultra-high-pressure liquid chromatography MS/MS coupled with quadrupole time-of-flight mass spectrometry. Finally, gene expression associated with CL biosynthesis and remodeling was quantified. Tissue histopathology and biochemical index evaluations revealed significant improvements in the liver after DR, except for the 60 DR group. The variation in mitochondrial CL distribution and DR levels showed an inverted U-shape, and the CL content in the 40 DR group was the most upregulated. This result is consistent with the results of the target metabolomic analysis, which showed that 40 DR presented more variation. Furthermore, DR led to increased gene expression associated with CL biosynthesis and remodeling. This study provides new insights into the mitochondrial mechanisms underlying DR intervention in obesity.

Insights into the Formation of Intermolecular Complexes of Fluorescent Probe 10-N-Nonyl Acridine Orange with Cardiolipin and Phosphatidylglycerol in Bacterial Plasma Membrane by Molecular Modeling.

In this article, we used molecular dynamics (MD), one of the most common methods for simulations of membranes, to study the interaction of fluorescent membranotropic biological probe 10-N-nonyl acridine orange (NAO) with the bilayer, mimicking a plasma membrane of Gram-negative bacteria. Fluorescent probes serve as an effective tool to study the localization of different components in biological membranes. Revealing the molecular details of their interaction with membrane phospholipids is important both for the interpretation of experimental results and future design of lipid-specific stains. By means of coarse-grained (CG) MD, we studied the interactions of NAO with a model membrane, imitating the plasma membrane of Gram-negative bacteria. In our simulations, we detected different NAO forms: monomers, dimers, and stacks. NAO dimers had the central cardiolipin (CL) molecule in a sandwich-like structure. The stacks were formed by NAO molecules interlayered with anionic lipids, predominantly CL. Use of the CG approach allowed to confirm the ability of NAO to bind to both major negatively charged phospholipids, phosphatidylglycerol (PG) and CL, and to shed light on the exact structure of previously proposed NAO-lipid complexes. Thus, CG modeling can be useful for the development of new effective and highly specific molecular probes.

Changes of mitochondrial lipid molecules, structure, cytochrome c and ROS of beef Longissimus lumborum and Psoas major during postmortem storage and their potential associations with beef quality.

This study investigated changes in mitochondrial lipid molecules and their potential associations with Longissimus lumborum (LL) and Psoas major (PM) quality during storage. A total of 1610 lipid species that matched with 36 lipid classes were identified from isolated mitochondria. PM had more key lipid molecules at storage d-1 including diacylglycerol and triacylglycerol (may play roles in membrane stability), phosphatidylethanolamine, acyl carnitine and cardiolipin (involved in energy metabolism), and cardiolipin and phosphatidylserine (important factors in apoptosis). Correspondingly, with extended storage time, mitochondrial structure, cytochrome c and reactive oxygen species (ROS) were changed, and muscle oxidation intensified. These changes have close associations with shear force and water holding capacity (WHC). Compared with LL, PM had higher content of lipid classes, more mitochondrial ROS, greater muscle oxidation, and lower shear force and WHC. These findings provided new insights into the effects of lipidome on mitochondrial structure, ROS and cytochrome c, and their potential associations with beef quality.

Phenotypic and genomic characteristics of Brevibacterium zhoupengii sp. nov., a novel halotolerant actinomycete isolated from bat feces.

Two strictly aerobic, Gram-staining-positive, non-spore-forming, regular rod-shaped (approximately 0.7 × 1.9 mm) bacteria (HY170T and HY001) were isolated from bat feces collected from Chongzuo city, Guangxi province (22°20'54″N, 106°49'20″E, July 2011) and Chuxiong Yi Autonomous Prefecture, Yunnan province (25°09'10″N, 102°04'39″E, October 2013) of South China, respectively. Optimal growth is obtained at 25-28°C (range, 4-32°C) on BHI-5% sheep blood plate with pH 7.5 (range, 5.0-10.0) in the presence of 0.5-1.0% NaCl (w/v) (range, 0-15% NaCl [w/v]). The phylogenetic and phylogenomic trees based respectively on the 16S rRNA gene and 845 core gene sequences revealed that the two strains formed a distinct lineage within the genus Brevibacterium, most closely related to B. aurantiacum NCDO 739T (16S rRNA similarity, both 98.5%; dDDH, 46.7-46.8%; ANI, 91.9-92.1%). Strain HY170T contained MK-8(H2), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG), galactose and ribose as the predominant menaquinone, major polar lipids, and main sugars in the cell wall teichoic acids, respectively. The meso-diaminopimelic acid (meso-DAP) was the diagnostic diamino acid of the peptidoglycan found in strain HY170T. Anteiso-C15:0 and anteiso-C17:0 were the major fatty acids (> 10%) of strains HY170T and HY001, with anteiso-C17:1A predominant in strain HY170T but absent in strain HY001. Mining the genomes revealed the presence of secondary metabolite biosynthesis gene clusters encoding for non-alpha poly-amino acids (NAPAA), ectoine, siderophore, and terpene. Based on results from the phylogenetic, chemotaxonomic and phenotypic analyses, the two strains could be classified as a novel species of the genus Brevibacterium, for which the name Brevibacterium zhoupengii sp. nov. is proposed (type strain HY170T = CGMCC 1.18600T = JCM 34230T).

Alteration of Mitochondrial Lipidome and Its Potential Effect on Apoptosis, Mitochondrial Reactive Oxygen Species Production, and Muscle Oxidation in Beef during Early Postmortem.

Lipid molecules are important participants in mitochondria-mediated apoptosis. This study explored the effect of mitochondrial lipids on mitochondria-mediated apoptosis, mitochondrial reactive oxygen species (ROS) production, and muscle oxidation of beef longissimus lumborum (LL, n = 6) and psoas major (PM, n = 6) during 24 h postmortem. A total of 432 lipid species matched with 21 lipid classes were identified. Remarkably, at 12 h postmortem, the levels of cardiolipin and phosphatidylserine in PM and ceramide, cardiolipin, phosphatidylserine, and sphingosine in LL increased significantly compared with 1 h postmortem, indicating that mitochondria-mediated apoptosis in beef muscle was accelerated during early postmortem. Moreover, PM had higher levels of cardiolipin and phosphatidylserine than LL, also suggesting a higher degree of apoptosis in PM during postmortem. Lipid molecules may assist the production of mitochondrial ROS and decrease the mitochondrial membrane potential (MMP) during postmortem apoptosis, resulting in muscle oxidation and the damage of antioxidant system. Notably, compared with LL, PM had higher abundance of apoptosis-related lipid molecules, a higher amount of ROS, faster diminishment in MMP, and then a higher degree of apoptosis. These findings provided new insights into the apoptosis and muscle biochemistry in beef during early postmortem.

Isolation and characterization of tick-borne Roseomonas haemaphysalidis sp. nov. and rodent-borne Roseomonas marmotae sp. nov.

Four novel Gram-negative, mesophilic, aerobic, motile, and cocci-shaped strains were isolated from tick samples (strains 546T and 573) and respiratory tracts of marmots (strains 1318T and 1311). The 16S rRNA gene sequencing revealed that strains 546T and 573 were 97.8% identical to Roseomonas wenyumeiae Z23T, whereas strains 1311 and 1318T were 98.3% identical to Roseomonas ludipueritiae DSM 14915T. In addition, a 98.0% identity was observed between strains 546T and 1318T. Phylogenetic and phylogenomic analyses revealed that strains 546T and 573 clustered with R. wenyumeiae Z23T, whereas strains 1311 and 1318T grouped with R. ludipueritiae DSM 14915T. The average nucleotide identity between our isolates and members of the genus Roseomonas was below 95%. The genomic G+C content of strains 546T and 1318T was 70.9% and 69.3%, respectively. Diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE) were the major polar lipids, with Q-10 as the predominant respiratory quinone. According to all genotypic, phenotypic, phylogenetic, and phylogenomic analyses, the four strains represent two novel species of the genus Roseomonas, for which the names Roseomonas haemaphysalidis sp. nov. and Roseomonas marmotae sp. nov. are proposed, with 546T (= GDMCC 1.1780T = JCM 34187T) and 1318T (= GDMCC 1.1781T = JCM 34188T) as type strains, respectively.

Host Gasdermin D restrains systemic endotoxemia by capturing Proteobacteria in the colon of high-fat diet-feeding mice.

Gasdermin D (GSDMD) functions as a key pyroptotic executor through its secreted N-terminal domain (GSDMD-N). However, the functional relevance and mechanistic basis of the precise roles of host colonic GSDMD in high-fat diet (HFD)-induced gut dysbiosis and systemic endotoxemia remain elusive. In this study, we demonstrate that HFD feeding triggers GSDMD-N secretion of both T-lymphocytes and enterocytes in mouse colons. GSDMD deficiency aggravates HFD-induced systemic endotoxemia, gut barrier impairment, and colonic inflammation. More importantly, active GSDMD-N kills the Proteobacteria phylum via directly interacting with Cardiolipin. Mechanistically, we identify that the Glu236 (a known residue for GSDMD protein cleavage) is a bona fide important site for the bacterial recognition of GSDMD. Collectively, our findings explain the mechanism by which colonic GSDMD-N maintains low levels of HFD-induced metabolic endotoxemia. A GSDMD-N mimetic containing an exposed Glu236 site could be an attractive strategy for the treatment of HFD-induced metabolic endotoxemia.

Tafazzin Deficiency Reduces Basal Insulin Secretion and Mitochondrial Function in Pancreatic Islets From Male Mice.

Tafazzin (TAZ) is a cardiolipin (CL) biosynthetic enzyme important for maintaining mitochondrial function. TAZ affects both the species and content of CL in the inner mitochondrial membrane, which are essential for normal cellular respiration. In pancreatic ß cells, mitochondrial function is closely associated with insulin secretion. However, the role of TAZ and CL in the secretion of insulin from pancreatic islets remains unknown. Male 4-month-old doxycycline-inducible TAZ knock-down (KD) mice and wild-type littermate controls were used. Immunohistochemistry was used to assess ß-cell morphology in whole pancreas sections, whereas ex vivo insulin secretion, CL content, RNA-sequencing analysis, and mitochondrial oxygen consumption were measured from isolated islet preparations. Ex vivo insulin secretion under nonstimulatory low-glucose concentrations was reduced ~52% from islets isolated from TAZ KD mice. Mitochondrial oxygen consumption under low-glucose conditions was also reduced ~58% in islets from TAZ KD animals. TAZ deficiency in pancreatic islets was associated with significant alteration in CL molecular species and elevated polyunsaturated fatty acid CL content. In addition, RNA-sequencing of isolated islets showed that TAZ KD increased expression of extracellular matrix genes, which are linked to pancreatic fibrosis, activated stellate cells, and impaired ß-cell function. These data indicate a novel role for TAZ in regulating pancreatic islet function, particularly under low-glucose conditions.

Streamlined Analysis of Cardiolipins in Prokaryotic and Eukaryotic Samples Using a Norharmane Matrix by MALDI-MSI.

Cardiolipins (CLs) are an important, regulated lipid class both in prokaryotic and eukaryotic cells, yet they remain largely unexplored by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in tissues. To date, no in-depth optimization studies of label-free visualization of CLs in complex biological samples have been reported. Here we report a streamlined modification to our previously reported MALDI-MSI method for detection of endogenous CLs in prokaryotic and eukaryotic cells based on preparation with norharmane (NRM) matrix. Notably, the use of NRM matrix permitted sensitive detection (4.7 pg/mm2) of spotted CL synthetic standards. By contrast, four other MALDI matrices commonly used for lipid analysis failed to generate CL ions. Using this NRM-based method, endogenous CLs were detected from two types of complex biological samples: dried bacterial arrays and mouse tissue sections. In both cases, using NRM resulted in a better signal/noise for CL ions than the other matrices. Furthermore, inclusion of a washing step improved CL detection from tissue and this combined tissue preparation method (washing and NRM matrix) was used to profile normal mouse lung. Mouse lung yielded 26 unique CLs that were mapped and identified. Consistent with previous findings, CLs containing polyunsaturated fatty acids (PUFAs) were found in abundance in the airway and vascular features of the lung. This work represents a comprehensive investigation of detection conditions for CL using MALDI-MSI in complex biological samples that resulted in a streamlined method that enables future studies of the biological role(s) of CL in tissue.

Proton Transfer Reactions for the Gas-Phase Separation, Concentration, and Identification of Cardiolipins.

Cardiolipin (CL) analysis demands high specificity, due to the extensive diversity of CL structures, and high sensitivity, due to their low relative abundance within the lipidome. While electrospray ionization mass spectrometry (ESI-MS) is the most widely used technology in lipidomics, the potential for multiple charging presents unique challenges for CL identification and quantification. Depending on the conditions, ESI-MS of lipid extracts in negative ion mode can give rise to cardiolipins ionized as both singly and doubly deprotonated anions. This signal degeneracy diminishes the signal-to-noise ratio, while in addition (for direct infusion), the dianion population falls within a m/z range already heavily congested with monoanions from more abundant glycerophospholipid subclasses. Herein, we describe a direct infusion strategy for CL profiling from total lipid extracts utilizing gas-phase proton-transfer ion/ion reactions. In this approach, lipid extracts are ionized by negative ion ESI generating both singly deprotonated phospholipids and doubly deprotonated CL anions. Charge reduction of the negative ion population by ion/ion reactions leads to an enhancement in singly deprotonated [CL - H]- species via proton transfer to the corresponding [CL - 2H]2-̅ dianions. To concentrate the [CL - H]- anion signal, multiple iterations of ion accumulation and proton-transfer ion/ion reaction can be performed prior to subsequent interrogation. Mass selection and collisional activation of the enriched population of [CL - H]- anions facilitates the assignment of individual fatty acyl substituents and phosphatidic acid moieties. Demonstrated advantages of this new approach derive from the improved performance in complex mixture analysis affording detailed characterization of low abundant CLs directly from a total biological extract.

The composition of phospholipid model bacterial membranes determines their endurance to secretory phospholipase A2 attack - The role of cardiolipin.

Soil bacteria are decomposer organisms crucial for the biodegradation of organic pollutants, mineralization of dead organic matter and the turnover of biogenic elements. In their environment they are constantly exposed to membrane-lytic enzymes emitted to the soil by other microorganisms competing for the same niche. Therefore, the composition and structure of their membranes is of utmost importance for survival in the harsh environment. Although soil bacteria species can be Gram-negative or Gram-positive and their membranes differ significantly, they are formed by phospholipids belonging mainly to three classes: phosphatidylethanolamines (PE), phosphatidylglycerols (PG) and cardiolipins (CL). The correlation of the membrane phospholipid composition and its susceptibility to secretory membrane-lytic enzymes is widely unknown; thus, to shed light on these phenomena we applied the Langmuir monolayer technique to construct models of soil bacteria membranes differing in the mutual proportion of the main phospholipids. To characterize the systems we studied their elasticity, mesoscopic texture, 2D crystalline structure and discussed the thermodynamics of the interactions between their components. The model membranes were exposed to secretory phospholipase A2. It turned out that in spite of the structural similarities the model membranes differed significantly in their susceptibility to s-PLA2 attack. The membranes devoid of cardiolipin were completely degraded, whereas, these containing cardiolipin were much more resistant to the enzymatic hydrolysis. It also turned out that the sole presence of cardiolipin in the model membrane did not guarantee the membrane durability and that the interplay between cardiolipin and the zwitterionic phosphatidylethanolamine was here of crucial importance.

Promotion of plasmalogen biosynthesis reverse lipid changes in a Barth Syndrome cell model.

In Barth syndrome (BTHS) mutations in tafazzin leads to changes in both the quantities and the molecular species of cardiolipin (CL), which are the hallmarks of BTHS. Contrary to the well-established alterations in CL associated with BTHS; recently a marked decrease in the plasmalogen levels in Barth specimens has been identified. To restore the plasmalogen levels, the present study reports the effect of promotion of plasmalogen biosynthesis on the lipidome of lymphoblasts derived from Barth patients as well as on cell viability, mitochondria biogenesis, and mitochondrial membrane potential. High resolution 31P NMR phospholipidomic analysis showed an increase in the levels of plasmenylethanolamine (the major plasmalogen in lymphoblasts), which reached values comparable to the control and a compensatory decrease in the levels of its diacyl-PE counterpart. Importantly, 31P NMR showed a significant increase in the levels of CL, while not altering the levels of monolysocardiolipin. Mass spectrometry measurements showed that the promotion of plasmalogen biosynthesis did not change the molecular species profile of targeted phospholipids. In addition, promotion of plasmalogen biosynthesis did not impact on cellular viability, although it significantly decrease mitochondria copy number and restored mitochondrial membrane potential. Overall, the results showed the efficacy of the promotion of plasmalogen biosynthesis on increasing the CL levels in a BTHS cell model and highlight the potential beneficial effect of a diet supplemented with plasmalogen precursors to BTHS patients.

Targeted lipidomics and transcriptomics profiling reveal the heterogeneity of visceral and subcutaneous white adipose tissue.

AIMS: The depot-specific differences in lipidome of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) reflect heterogeneity of white adipose tissue (WAT), which plays a central role in its distinct response to outside stimuli. However, the detailed lipidome of depot-specific WAT is largely unknown, especially the minor constitutes including phospholipid and sphingolipid. MATERIALS AND METHODS: To investigate this field, we applied a high-coverage targeted lipidomics approach of VAT and SAT in male C57BL/6J mice to compare the basal level of their lipid profiles. Applying microarray and quantitative real-time polymerase chain reaction, we analyzed the transcriptome of twodepot-specific WAT and verified the differences in individual genes. KEY FINDINGS: In total, 342 lipid species from 19 lipid classes were identified. Our results showed the composition of TAG and FFA were different in length of chain and saturation. Interestingly, low abundance phospholipid, sphingolipid and cardiolipin were significantly higher in SAT. Lipid correlation network analysis vindicated that TAG and phospholipid formed distinct subnet and had more connections with other lipid species. Enriched ontology analysis of gene screened from LIPID MAPS and microarray suggested the differences were mainly involved in lipid metabolism, insulin resistance and inflammatory response. SIGNIFICANCE: Our comprehensive lipidomics and transcriptomics analyses revealed differences in lipid composition and lipid metabolism of two depot-specific WAT, which would offer new insights into the investigation of heterogeneity of visceral and subcutaneous white adipose tissue.

Mass spectrometric investigation of cardiolipins and their oxidation products after two-dimensional heart-cut liquid chromatography.

The anionic phospholipid class of cardiolipins (CL) is increasingly attracting scientific attention in the recent years. CL can be found as a functional component of mitochondrial membranes in almost all living organisms. Changes in the CL composition are favored by oxidative stress. Based on this finding, the investigation of CL and their oxidation products in relation to various disease patterns, including neurodegenerative ones, is moving into the focus of current research. The analysis of this diverse lipid class is still challenging and requires sensitive and selective methods. In this work, we demonstrate an online two-dimensional liquid chromatography (2D-LC) approach by means of a heart-cut setup. In the first dimension, a fast hydrophilic interaction liquid chromatography (HILIC) method was developed for the separation of CL and their oxidation products from other phospholipid classes, but more important from nonpolar lipid classes, such as triacylglycerol and cholesterol. Those classes can negatively affect the electrospray ionization and also the chromatography. For the heart-cut approach, the CL fraction was selectively transferred to a loop using a six-port valve followed by the transfer to a reversed phase (RP) column in second dimension. On the RP column, the transferred CL fraction including the oxidation products were separated according to the hydrophobicity of acyl chain moieties. Matrix effects were significantly reduced compared to the one-dimensional LC-MS method. In addition, the total separation time had not to be prolonged by shifting the equilibration step of the RP column parallel to the separation in first dimension. The heart-cut LC-LC approach was applied to artificially oxidized lipid extracts of bovine heart and yeast by means of Fenton reaction. In summary, 42 species have been identified by high resolution mass spectrometry and database matching. 31 species thereof have been further characterized by MS/MS experiments.

Quantifying Lipid Mobility and Peptide Binding for Gram-Negative and Gram-Positive Model Supported Lipid Bilayers.

Model membranes are a valuable tool to investigate the mechanism of interaction between antibiotic compounds and bacterial membranes. However, the development of supported lipid bilayer (SLB) models for Gram-negative and Gram-positive bacteria has been challenging because of the high charge and spontaneous curvature of the lipids that make up these membranes. Here we describe a method for preparing mimetic Gram-negative inner membrane and Gram-positive membrane SLBs, including asymmetric SLBs (asy-SLBs) that contain a fluorescent tracer only in the upper leaflet of the membrane. We quantified the dynamics of the lipids in these membranes with fluorescence correlation spectroscopy (FCS) and found that lipid diffusion is slower in Gram-negative SLBs/asySLBs than in Gram-positive SLBs/asySLBs. Peptide binding to these membranes was also characterized using colistin, a Gram-negative specific antibiotic. Interactions between colistin and membrane lipids phosphatidylethanolamine (PE) or cardiolipin (TOCL) were probed with pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Overall, our data provide unique insight into the diffusion dynamics of lipids in Gram-negative and Gram-positive membranes as well as a novel platform for investigating the mechanism of interaction between antibiotic peptides and bacterial membrane lipids.

Determination of the collision cross sections of cardiolipins and phospholipids from Pseudomonas aeruginosa by traveling wave ion mobility spectrometry-mass spectrometry using a novel correction strategy.

Collision cross section (CCS) values are descriptors of the 3D structure of ions which can be determined by ion mobility spectrometry (IMS). Currently, most lipidomic studies involving CCS value determination concern eukaryote samples (e.g. human, bovine) and to a lower extent prokaryote samples (e.g. bacteria). Here, we report CCS values obtained from traveling wave ion mobility spectrometry (TWCCSN2) measurements from the bacterial membrane of Pseudomonas aeruginosa-a bacterium ranked as priority 1 for the R&D of new antibiotics by the World Health Organization. In order to cover the lack of reference compounds which could cover the m/z and CCS ranges of the membrane lipids of P. aeruginosa, three calibrants (polyalanine, dextran and phospholipids) were used for the TWCCSN2 calibration. A shift from the published lipid CCS values was systematically observed (ΔCCS% up to 9%); thus, we proposed a CCS correction strategy. This correction strategy allowed a reduction in the shift (ΔCCS%) between our measurements and published values to less than 2%. This correction was then applied to determine the CCS values of Pseudomonas aeruginosa lipids which have not been published yet. As a result, 32 TWCCSN2 values for [M+H]+ ions and 24 TWCCSN2 values for [M-H]- ions were obtained for four classes of phospholipids (phosphatidylethanolamines (PE), phosphatidylcholines (PC), phosphatidylglycerols (PG) and diphosphatidylglycerols-known as cardiolipins (CL)). Graphical abstract.