A Robust Lipidomics Workflow for Mammalian Cells, Plasma, and Tissue Using Liquid-Chromatography High-Resolution Tandem Mass Spectrometry

et al. 2020

Sci Rep

Lipoyl synthases are key enzymes in lipoic acid biosynthesis, a co-factor of several enzyme complexes involved in central metabolism. Plant pyruvate dehydrogenase complex (PDH), located in mitochondria and plastids, catalyses the first step of fatty acid biosynthesis in these organelles. Among their different components, the E2 subunit requires the lipoic acid prosthetic group to be active. De novo lipoic acid biosynthesis is achieved by the successive action of two enzymes on octanoyl-ACP: octanoyltransferase (LIP2) and lipoyl synthase (LIP1). In this study, two plastidial lipoyl synthase genes from sunflower (Helianthus annuus L.) were identified (HaLIP1p1 and HaLIP1p2), sequenced and cloned in a heterologous production system (Escherichia coli). Gene expression studies revealed similar expression patterns for both isoforms, with a slight predominance of HaLIP1p1 in vegetative tissues and mature seeds. Tertiary structural models for these enzymes indicate they both have the same theoretical catalytic sites, using lipoyl-lys and 5-deoxyadenosine as docking substrates. The fatty acid profile of E. coli cells overexpressing HaLIP1p1 and HaLIP1p2 did not present major differences, and the in vivo activity of both proteins was confirmed by complementation of an E. coli JW0623 mutant in which lipoyl synthase is defective. Although no significant differences were detected in the total fatty acid composition of transgenic Arabidopsis thaliana seeds overexpressing any of both proteins, a lipidomic analysis revealed a redistribution of the glycerolipid species, accompanied with increased phosphatidylethanolamine (PE) content and a decrease in diacyglycerols (DAG) and phosphatidylcholine (PC). Depletion of the SAM co-factor caused by HaLIP1p1 and HaLIP1p2 overexpression in transgenic plants could explain this remodelling through its effects on PC synthesis. De novo fatty acid synthesis in plants takes place in the plastid, with acetyl-coenzyme A (acetyl-CoA) the main precursor. This metabolite is primarily generated from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO 2 and NADH 1. The PDH complex contains 3 subunits: E1 or pyruvate dehydrogenase, E2 or dihydrolipoyl acetyltransferase, and E3 or dihydrolipoyl transhydrogenase. Of these, E2 requires a lipoic acid (LA; 6,8-dithiooctanoic acid or 1,2-dithiolane-3-pentanoic acid) prosthetic group to be functional, a sulphur-containing enzyme co-factor that is essential for the catalytic activity of several key enzyme complexes involved in fundamental metabolic processes in most prokaryotic and eukaryotic organisms. Besides PDH, this prosthetic group is also required by α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, acetoin dehydrogenase, and the glycine cleavage complex (glycine decarboxylase) 2-5 .

Section: Generation Of Transgenic Plants and Vector Constructionsmentioning

confidence: 99%

Impact of sunflower (Helianthus annuus L.) plastidial lipoyl synthases genes expression in glycerolipids composition of transgenic Arabidopsis plants

Martins-Noguerol

Moreno‐Pérez

et al. 2020

Sci Rep

“…The lipids were resuspended in 200 μl of isopropanol. LC was performed based on a modified protocol from reference 71 . Briefly, a high-performance liquid chromatograph (HPLC) 1290 (Agilent Technologies) with a C 18 Hypersil gold column (100 mm by 2.1 mm, 1.9 μm; Thermo Fisher) at 50°C was used.…”

Section: Methodsmentioning

confidence: 99%

The Impact of Plasma Membrane Lipid Composition on Flagellum-Mediated Adhesion of Enterohemorrhagic Escherichia coli

Cazzola

Lemaire

et al. 2020

mSphere

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell’s plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions. IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

“…The lipids were resuspended in 200 µl of isopropanol. LC was performed based on a modified protocol from (65). Briefly an HPLC 1290 (Agilent Technologies) with a C18 Hypersil Gold (100 x 2.1 mm, 1.9 μm, Thermofisher) at 50°C was used.…”

Section: Methodsmentioning

confidence: 99%

The impact of plasma membrane lipid composition on flagella-mediated adhesion of enterohemorrhagicEscherichia coli

Cazzola

Lemaire

et al. 2020

Preprint

AbstractEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC on host tissues is the first step enabling bacterial colonization. Adhesins like fimbriae and flagella mediate this mechanism. Here, we studied the interaction of the bacterial flagellum with the host cell’s plasma membrane using Giant Unilamellar Vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components including proteins and glycoproteins. In contrast, with GUVs we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild type EHEC but not of a non-flagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagella-mediated adhesion to plasma membranes has broad implications to host-pathogen interactions.ImportanceBacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues and form biofilm. Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagella-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagella adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts are important in bacterial adhesion. These findings significantly strengthen plasma membrane adhesion via bacterial flagella in an important human pathogen. This mechanism represents a promising target for the development of novel anti-adhesion therapies.