Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis

ABSTRACT Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium discoideum are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, its natural food substrate.

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A lipid droplet-peroxisome network drives longevity by monounsaturated fatty acids via modulating ether lipid synthesis and ferroptosis

10.21203/rs.3.rs-740521/v1 ◽

2021 ◽

Author(s):

Anne Brunet ◽

Katharina Papsdorf ◽

Amir Hosseini ◽

Jason Miklas ◽

Matias Cabruja ◽

...

Keyword(s):

Fatty Acids ◽

Lipid Droplet ◽

Lipid Droplets ◽

Unsaturated Fatty Acids ◽

Lipid Synthesis ◽

Ether Lipid ◽

Monounsaturated Fatty Acids ◽

Human Longevity ◽

Ether Lipids ◽

C Elegans

Abstract Dietary mono-unsaturated fatty acids (MUFAs) are linked to human longevity and extend lifespan in several species1-12. But the mechanisms by which MUFAs promote longevity remain unclear. Here we show that an organelle hub involving lipid droplets and peroxisomes is critical for lifespan extension by MUFAs in C. elegans. MUFA accumulation increases lipid droplet number in fat storage tissues, and lipid droplet synthesis is necessary for MUFA-mediated longevity. Interestingly, the number of lipid droplets in young individuals can predict their remaining lifespan. MUFA accumulation also increases the number of peroxisomes, and peroxisome activity is required for MUFA-mediated longevity. By performing a targeted screen, we uncover a functional network between lipid droplets and peroxisomes in longevity. Interestingly, our screen also identifies ether lipids as critical components of the lipid droplet-peroxisome network. Using lipidomics, we find that the ratio of MUFAs to polyunsaturated fatty acids (PUFAs) in ether lipids is increased by MUFA accumulation. Ether lipids are involved in ferroptosis, a non-apoptotic form of cell death13-17, and MUFAs promote longevity in part via suppression of ferroptosis. Our results identify a mechanism of action for MUFAs to extend lifespan and uncover an organelle network involved in the homeostasis of MUFA-rich ether lipids. Our work also opens new avenues for lipid-based interventions to delay aging.

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Two DHH Subfamily 1 Proteins Contribute to Pneumococcal Virulence and Confer Protection against Pneumococcal Disease

Infection and Immunity ◽

10.1128/iai.01383-10 ◽

2011 ◽

Vol 79 (9) ◽

pp. 3697-3710 ◽

Cited By ~ 28

Author(s):

L. E. Cron ◽

K. Stol ◽

P. Burghout ◽

S. van Selm ◽

E. R. Simonetti ◽

...

Keyword(s):

Otitis Media ◽

Pneumococcal Disease ◽

Capsular Polysaccharide ◽

Pneumococcal Infection ◽

Bacterial Pathogen ◽

Protein Vaccine ◽

Double Knockout ◽

Content Type ◽

Knockout Mutants ◽

Different Strains

ABSTRACTStreptococcus pneumoniaeis an important human bacterial pathogen, causing such infections as pneumonia, meningitis, septicemia, and otitis media. Current capsular polysaccharide-based conjugate vaccines protect against a fraction of the over 90 serotypes known, whereas vaccines based on conserved pneumococcal proteins are considered promising broad-range alternatives. The pneumococcal genome encodes two conserved proteins of an as yet unknown function, SP1298 and SP2205, classified as DHH (Asp-His-His) subfamily 1 proteins. Here we examined their contribution to pneumococcal pathogenesis using single and double knockout mutants in three different strains: D39, TIGR4, and BHN100. Mutants lacking both SP1298 and SP2205 were severely impaired in adherence to human epithelial Detroit 562 cells. Importantly, the attenuated phenotypes were restored upon genetic complementation of the deleted genes. Single and mixed mouse models of colonization, otitis media, pneumonia, and bacteremia showed that bacterial loads in the nasopharynx, middle ears, lungs, and blood of mice infected with the mutants were significantly reduced from those of wild-type-infected mice, with an apparent additive effect upon deletion of both genes. Minor strain-specific phenotypes were observed, i.e., deletion of SP1298 affected host-cell adherence in BHN100 only, and deletion of SP2205 significantly attenuated virulence in lungs and blood in D39 and BHN100 but not TIGR4. Finally, subcutaneous vaccination with a combination of both DHH subfamily 1 proteins conferred protection to nasopharynx, lungs, and blood of mice infected with TIGR4. We conclude that SP1298 and SP2205 play a significant role at several stages of pneumococcal infection, and importantly, these proteins are potential candidates for a multicomponent protein vaccine.

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Addition of lipid substituents of mammalian protein glycosylphosphoinositol anchors

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.21-31.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 21-31

Author(s):

N Singh ◽

R A Zoeller ◽

M L Tykocinski ◽

P B Lazarow ◽

A M Tartakoff

Keyword(s):

Lipid Synthesis ◽

Ether Lipid ◽

Raw 264.7 ◽

Placental Alkaline Phosphatase ◽

Ether Lipids ◽

Wild Type ◽

Animal Cells ◽

Express Cell ◽

Mutant Cells ◽

Mammalian Protein

A single metabolic path leading to synthesis of ether lipids is known in animal cells, the major products of which are plasmalogens. To learn whether this peroxisomal path is also responsible for the synthesis of base-resistant lipid components of glycosylphosphoinositol (GPI)-anchored membrane proteins, we have investigated the structure of anchor precursor mannolipids both in wild-type cells (CHO-K1 and a macrophage-like line, RAW 264.7) and in two corresponding mutant cells in which ether lipid biosynthesis is severely impaired. We observe that the precursor mannolipids of both the wild-type and mutant cells do not include alkylglycerol. Nevertheless, both wild-type and mutant cells express cell surface GPI-anchored placental alkaline phosphatase (AP) which includes alkali-resistant hydrophobic chains in its anchor moiety. Thus, (i) in normal AP GPI anchor synthesis, any ether-linked substituents must be added either immediately before, during, or after anchor addition to AP, and (ii) the classical peroxisomal path for ether lipid synthesis appears not to contribute to the synthesis of GPI anchors.

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Development of a Markerless Knockout Method for Actinobacillus succinogenes

Applied and Environmental Microbiology ◽

10.1128/aem.00492-14 ◽

2014 ◽

Vol 80 (10) ◽

pp. 3053-3061 ◽

Cited By ~ 11

Author(s):

Rajasi V. Joshi ◽

Bryan D. Schindler ◽

Nikolas R. McPherson ◽

Kanupriya Tiwari ◽

Claire Vieille

Keyword(s):

High Efficiency ◽

Direct Selection ◽

Selection Marker ◽

Actinobacillus Succinogenes ◽

Knockout Mutant ◽

Double Knockout ◽

Pyruvate Formate Lyase ◽

Content Type ◽

Knockout Mutants ◽

Encoding Genes

ABSTRACTActinobacillus succinogenesis one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method forA. succinogenesusing natural transformation or electroporation. TheEscherichia coliisocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use ofA. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. TheSaccharomyces cerevisiaeflippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressingflpwas cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

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Binding of Complement Factor H to PorB3 and NspA Enhances Resistance of Neisseria meningitidis to Anti-Factor H Binding Protein Bactericidal Activity

Infection and Immunity ◽

10.1128/iai.02984-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1536-1545 ◽

Cited By ~ 25

Author(s):

Serena Giuntini ◽

Rolando Pajon ◽

Sanjay Ram ◽

Dan M. Granoff

Keyword(s):

Neisseria Meningitidis ◽

Bactericidal Activity ◽

Binding Protein ◽

Factor H ◽

Resistant Isolate ◽

Complement Factor ◽

Double Knockout ◽

Content Type ◽

Knockout Mutants ◽

Sensitive Isolate

Among 25 serogroup BNeisseria meningitidisclinical isolates, we identified four (16%) with high factor H binding protein (FHbp) expression that were resistant to complement-mediated bactericidal activity of sera from mice immunized with recombinant FHbp vaccines. Two of the four isolates had evidence of human FH-dependent complement downregulation independent of FHbp. Since alternative complement pathway recruitment is critical for anti-FHbp bactericidal activity, we hypothesized that in these two isolates binding of FH to ligands other than FHbp contributes to anti-FHbp bactericidal resistance. Knocking out NspA, a known meningococcal FH ligand, converted both resistant isolates to anti-FHbp susceptible isolates. The addition of a nonbactericidal anti-NspA monoclonal antibody to the bactericidal reaction also increased anti-FHbp bactericidal activity. To identify a role for FH ligands other than NspA or FHbp in resistance, we created double NspA/FHbp knockout mutants. Mutants from both resistant isolates bound 10-fold more recombinant human FH domains 6 and 7 fused to Fc than double knockout mutants prepared from two sensitive meningococcal isolates. In light of recent studies showing functional FH-PorB2 interactions, we hypothesized that PorB3 from the resistant isolates recruited FH. Allelic exchange ofporB3from a resistant isolate to a sensitive isolate increased resistance of the sensitive isolate to anti-FHbp bactericidal activity (and vice versa). Thus, some PorB3 variants functionally bind human FH, which in the presence of NspA enhances anti-FHbp resistance. Combining anti-NspA antibodies with anti-FHbp antibodies can overcome resistance. Meningococcal vaccines that target both NspA and FHbp are likely to confer greater protection than either antigen alone.

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Conserved function of ether lipids and sphingolipids in the early secretory pathway

10.1101/2019.12.19.881094 ◽

2019 ◽

Author(s):

Noemi Jiménez-Rojo ◽

Manuel D. Leonetti ◽

Valeria Zoni ◽

Adai Colom ◽

Suihan Feng ◽

...

Keyword(s):

Membrane Trafficking ◽

Cell Biology ◽

Secretory Pathway ◽

Protein Transport ◽

Lipid Synthesis ◽

Chemical Properties ◽

Ether Lipid ◽

Ether Lipids ◽

Early Secretory Pathway ◽

A Genome

ABSTRACTSphingolipids have been shown to play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, where depletion of one of these lipid types resulted in increases in the other, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as GPI anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.

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Addition of lipid substituents of mammalian protein glycosylphosphoinositol anchors.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.21 ◽

1994 ◽

Vol 14 (1) ◽

pp. 21-31 ◽

Cited By ~ 15

Author(s):

N Singh ◽

R A Zoeller ◽

M L Tykocinski ◽

P B Lazarow ◽

A M Tartakoff

Keyword(s):

Lipid Synthesis ◽

Ether Lipid ◽

Raw 264.7 ◽

Placental Alkaline Phosphatase ◽

Ether Lipids ◽

Wild Type ◽

Animal Cells ◽

Express Cell ◽

Mutant Cells ◽

Mammalian Protein

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Bioactive Ether Lipids: Primordial Modulators of Cellular Signaling

Metabolites ◽

10.3390/metabo11010041 ◽

2021 ◽

Vol 11 (1) ◽

pp. 41

Author(s):

Nikhil Rangholia ◽

Tina M. Leisner ◽

Stephen P. Holly

Keyword(s):

Mammalian Cells ◽

Ether Lipid ◽

Cell Surface Receptor ◽

Ether Lipids ◽

Permeable Barrier ◽

Essential Components ◽

Intracellular Regulation ◽

Enzymatic Cascades ◽

Surface Receptor ◽

Functional Mechanisms

The primacy of lipids as essential components of cellular membranes is conserved across taxonomic domains. In addition to this crucial role as a semi-permeable barrier, lipids are also increasingly recognized as important signaling molecules with diverse functional mechanisms ranging from cell surface receptor binding to the intracellular regulation of enzymatic cascades. In this review, we focus on ether lipids, an ancient family of lipids having ether-linked structures that chemically differ from their more prevalent acyl relatives. In particular, we examine ether lipid biosynthesis in the peroxisome of mammalian cells, the roles of selected glycerolipids and glycerophospholipids in signal transduction in both prokaryotes and eukaryotes, and finally, the potential therapeutic contributions of synthetic ether lipids to the treatment of cancer.

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The carmine dilemma: does the natural colourant preference outweigh nausea?

British Food Journal ◽

10.1108/bfj-12-2017-0671 ◽

2018 ◽

Vol 120 (8) ◽

pp. 1915-1928

Author(s):

Judith Müller-Maatsch ◽

Johannes Jasny ◽

Katharina Henn ◽

Claudia Gras ◽

Reinhold Carle

Keyword(s):

Design Methodology ◽

Consumer Perception ◽

Natural Food ◽

German Population ◽

Misleading Information ◽

Content Type ◽

Broad Cross Section ◽

First Time ◽

Self Administered Questionnaire ◽

Insight Into

Purpose The purpose of this paper is to provide insight into the consumers’ perception of natural and artificial food colourants. Furthermore, attitudes towards the application of carmine, being technically important and ubiquitously used to impart red shades, are assessed and analysed. Originating from insects, carmine is considered as natural but may arouse disgust. Design/methodology/approach In total, 625 individuals were surveyed using an online, self-administered questionnaire to represent a broad cross-section of the German population. Findings Independent of their origin, the application of colourants was rejected by 57.0 per cent of the interviewees. In total, 31.8 per cent of the participants stated a neutral attitude, while only 11.2 per cent expressed a positive notion. Most respondents preferred colourants from natural sources to artificial ones. While consumers perceive natural food colourants composed of genuine plant pigments positively, 61.6 per cent of respondents disliked the application of animal-derived colourants, 24.8 per cent of them did neither reject nor like it, and only 13.6 per cent of the interviewees stated a positive attitude towards them. The findings of this paper further indicate consumers’ preference for colourants to be either artificial or plant-derived rather than carmine. Food colourants are being rejected, possibly due to misleading information and confusing labelling. Consequently, information about carmine, including its origin and production, did not increase the aversion to products that are dyed with it, but increased their acceptance. Originality/value This study outlines consumer perception and attitudes towards food colourants. For the first time, the findings of this paper report the effect of revealing information about an additive, which initially aroused disgust, and its influence on consumer perception.

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A Distinctive and Host-Restricted Gut Microbiota in Populations of a Cactophilic Drosophila Species

Applied and Environmental Microbiology ◽

10.1128/aem.01551-17 ◽

2017 ◽

Vol 83 (23) ◽

Cited By ~ 22

Author(s):

Vincent G. Martinson ◽

Javier Carpinteyro-Ponce ◽

Nancy A. Moran ◽

Therese A. Markow

Keyword(s):

Sonoran Desert ◽

Natural Habitat ◽

Natural Food ◽

Content Type ◽

Columnar Cacti ◽

Social Bees ◽

Insect Gut ◽

Almost All ◽

Natural Settings ◽

Bacterial Groups

ABSTRACT Almost all animals possess gut microbial communities, but the nature of these communities varies immensely. For example, in social bees and mammals, the composition is relatively constant within species and is dominated by specialist bacteria that do not live elsewhere; in laboratory studies and field surveys of Drosophila melanogaster, however, gut communities consist of bacteria that are ingested with food and that vary widely among individuals and localities. We addressed whether an ecological specialist in its natural habitat has a microbiota dominated by gut specialists or by environmental bacteria. Drosophila nigrospiracula is a species that is endemic to the Sonoran Desert and is restricted to decaying tissues of two giant columnar cacti, Pachycereus pringlei (cardón cactus) and Carnegiea gigantea (saguaro cactus). We found that the D. nigrospiracula microbiota differs strikingly from that of the cactus tissue on which the flies feed. The most abundant bacteria in the flies are rare or completely absent in the cactus tissue and are consistently abundant in flies from different cacti and localities. Several of these fly-associated bacterial groups, such as the bacterial order Orbales and the genera Serpens and Dysgonomonas, have been identified in prior surveys of insects from the orders Hymenoptera, Coleoptera, Lepidoptera, and Diptera, including several Drosophila species. Although the functions of these bacterial groups are mostly unexplored, Orbales species studied in bees are known to break down plant polysaccharides and use the resulting sugars. Thus, these bacterial groups appear to be specialized to the insect gut environment, where they may colonize through direct host-to-host transmission in natural settings. IMPORTANCE Flies in the genus Drosophila have become laboratory models for microbiota research, yet the bacteria commonly used in these experiments are rarely found in wild-caught flies and instead represent bacteria also present in the food. This study shows that an ecologically specialized Drosophila species possesses a distinctive microbiome, composed of bacterial types absent from the flies' natural food but widespread in other wild-caught insects. This study highlights the importance of fieldwork-informed microbiota research.

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