Markus Maniak

Ether lipids in varying amounts are membrane constituents and storage material in the protist and animal kingdoms, but are largely absent from fungi and plants. Their biosynthesis pathway starts in the peroxisome and involves a set of well-conserved enzymes. Only one step, the reduction of alkyl-dihydroxyacetone-phosphate to alkyl-glycerol-3-phosphate, is mediated by so-called short-chain dehydrogenases/reductases, which are members of huge protein families. Here, using GFP fusions, we identify a peroxisomal enzyme in Dictyostelium, as well as a highly related protein residing in the endoplasmic reticulum. Single-gene knockouts indicate that these enzymes largely compensate for one another, suggesting a flexible redistribution of lipid metabolites between these organelles. The double knockout, however, is severely affected in ether lipid composition and displays a clear growth retardation. The defects can also be reverted by expression of the cognate yeast enzyme, demonstrating conservation of this metabolic step across kingdoms of life.

Dictyostelium amoebae store surplus fatty acids from the diet in form of lipid droplets. Some of the enzymes governing neutral lipid synthesis are already known. For the phosphatidic acid-specific phosphatases, six genes were found, one of which was automatically annotated as LPIN2. Two GFP-tagged variants of LPIN2 homogeneously distribute in the cytoplasm and no organelle association was observed. LPIN2 ^– mutants contain less than 17% residual amount of the major neutral lipid species, but phospholipid amounts are not obviously affected. A growth retardation on bacteria as food source may suggest that lipid droplets serve to detoxify excess free fatty acids.

Lipids are the building blocks for cellular membranes; they provide signalling molecules for membrane dynamics and serve as energy stores. One path of their synthesis is initiated by glycerol-3-phosphate acyltransferase (GPAT), which in Dictyostelium resides on the endoplasmic reticulum. When an excess of fatty acids is present, it redistributes to storage organelles, the lipid droplets. Mutants, where the GPAT was eliminated by homologous recombination, produce fewer lipid droplets and are almost devoid of triacylglycerols (TAG), rendering them more resistant to cell death and cell loss in the developmental stages preceding fruiting body formation. The enzyme most closely related to GPAT is called FARAT, because it combines a fatty acyl-reductase (FAR) and an acyltransferase (AT) domain in its sequence. The protein is confined to the lumen of the peroxisome, where it transfers a fatty acid to dihydroxyacetone-phosphate initiating the synthesis of ether lipids, later completed at the endoplasmic reticulum. A mutant lacking FARAT produces lipid droplets that are devoid of the storage lipid monoalkyl-diacyl-glycerol (MDG), but the efficiency of spore formation in the developmental cycle is largely unaltered. Instead, these mutants are strongly impaired in phagocytosis of yeast particles, which is attributed to reduced synthesis of membrane phospholipids containing ether-linked chains.

Triacylglycerol is a universal storage molecule for metabolic energy in living organisms. However, Dictyostelium amoebae, that have accumulated storage fat from added fatty acids do not progress through the starvation period preceding the development of the durable spore. Mutants deficient in genes of fat metabolism, such as fcsA, encoding a fatty acid activating enzyme, or dgat1 and dgat2, specifying proteins that synthesize triacylglycerol, strongly increase their chances to contribute to the spore fraction of the developing fruiting body, but lose the ability to produce storage fat efficiently. Dictyostelium seipin, an orthologue of a human protein that in patients causes the complete loss of adipose tissue when mutated, does not quantitatively affect fat storage in the amoeba. Dictyostelium seiP knockout mutants have lipid droplets that are enlarged in size but reduced in number. These mutants are as vulnerable as the wild type when exposed to fatty acids during their vegetative growth phase, and do not efficiently enter the spore head in Dictyostelium development.

Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from Dictyostelium discoideum, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for TspA-E in the multicellular slug stage, whereas vegetative cells expressed only TspA, TspC and, to a lesser extent, TspD. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. N-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC in vivo. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of TspC and TspD exibited unaltered growth, adhesion, random motility and development. Yet, tspC⁻ cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of TspC rescued their phenotype. In conclusion, our data fill a gap in Dictyostelium research and open up the possibility that Tsps in contractile vacuoles of e.g. Trypanosoma may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases.

Lipid droplets exist in virtually every cell type, ranging not only from mammals to plants, but also to eukaryotic and prokaryotic unicellular organisms such as Dictyostelium and bacteria. They serve among other roles as energy reservoir that cells consume in times of starvation. Mycobacteria and some other intracellular pathogens hijack these organelles as a nutrient source and to build up their own lipid inclusions. The mechanisms by which host lipid droplets are captured by the pathogenic bacteria are extremely poorly understood. Using the powerful Dictyostelium discoideum/Mycobacterium marinum infection model, we observed that, immediately after their uptake, lipid droplets translocate to the vicinity of the vacuole containing live but not dead mycobacteria. Induction of lipid droplets in Dictyostelium prior to infection resulted in a vast accumulation of neutral lipids and sterols inside the bacterium-containing compartment. Subsequently, under these conditions, mycobacteria accumulated much larger lipid inclusions. Strikingly, the Dictyostelium homologue of perilipin and the murine perilipin 2 surrounded bacteria that had escaped to the cytosol of Dictyostelium or microglial BV-2 cells respectively. Moreover, bacterial growth was inhibited in Dictyostelium plnA knockout cells. In summary, our results provide evidence that mycobacteria actively manipulate the lipid metabolism of the host from very early infection stages.

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.

Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general, it consists of a hydrophobic core of triglycerides and steryl esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here, we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel protein components are LdpA, a protein specific to Dictyostelium , and Net4, which has strong homologies to mammalian DUF829/Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane.