Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase

The obligate intracellular parasite Chlamydia trachomatis has a reduced genome but relies on de novo fatty acid and phospholipid biosynthesis to produce its membrane phospholipids. Lipidomic analyses showed that 8% of the phospholipid molecular species synthesized by C. trachomatis contained oleic acid, an abundant host fatty acid that cannot be made by the bacterium. Mass tracing experiments showed that isotopically labeled palmitic, myristic, and lauric acids added to the medium were incorporated into C. trachomatis-derived phospholipid molecular species. HeLa cells did not elongate lauric acid, but infected HeLa cell cultures elongated laurate to myristate and palmitate. The elongated fatty acids were incorporated exclusively into C. trachomatis-produced phospholipid molecular species. C. trachomatis has adjacent genes encoding the separate domains of the bifunctional acyl-acyl carrier protein (ACP) synthetase/2-acylglycerolphosphoethanolamine acyltransferase gene (aas) of Escherichia coli. The CT775 gene encodes an acyltransferase (LpaT) that selectively transfers fatty acids from acyl-ACP to the 1-position of 2-acyl-glycerophospholipids. The CT776 gene encodes an acyl-ACP synthetase (AasC) with a substrate preference for palmitic compared with oleic acid in vitro. Exogenous fatty acids were elongated and incorporated into phospholipids by Escherichia coli-expressing AasC, illustrating its function as an acyl-ACP synthetase in vivo. These data point to an AasC-dependent pathway in C. trachomatis that selectively scavenges host saturated fatty acids to be used for the de novo synthesis of its membrane constituents.     

Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC₅₀ of 0.9 μm at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.     

Lipids of fish parasites and their hosts: fatty acids of phospholipids of Paratenuisentis ambiguus and its host eel (Anguilla anguilla)

The fatty acid composition of various phospholipid classes, e.g. phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and sphingomyelins, of the fish endoparasite Paratenuisentis ambiguus and of intestinal tissue of its host, eel (Anguilla anguilla), were investigated. Phospholipids from parasite and infected host intestine show considerable differences in the fatty acid compositions. High proportions of saturated long-chain and very long-chain acyl moieties were found in phosphatidylcholines, ethanolamines and serines of the parasite. Membrane phospholipids of P. ambiguus contain remarkably high proportions of polyunsaturated acyl moieties, in particular eicosapentaenoyl moieties (20:5 n-3), which undoubtedly originate from aquatic feed or host intestine. It is suggested that the parasite's membranes are stabilized by incorporation of high proportions of saturated long-chain and very long-chain fatty acids to counterbalance the destabilizing effects of the polyunsaturated fatty acids.     

Host HDL biogenesis machinery is recruited to the inclusion of Chlamydia trachomatis-infected cells and regulates chlamydial growth

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that is the most common cause of sexually transmitted bacterial infections and is the etiological agent of trachoma, the leading cause of preventable blindness. The organism infects epithelial cells of the genital tract and eyelid resulting in a damaging inflammatory response. Chlamydia trachomatis grows within a vacuole termed the inclusion, and its growth depends on numerous host factors, including lipids. Although a variety of mechanisms are involved in the acquisition of host cell cholesterol and glycosphingolipids by C. trachomatis, none of the previously documented pathways for lipid acquisition are absolutely required for growth. Here we demonstrate that multiple components of the host high‐density lipoprotein (HDL) biogenesis machinery including the lipid effluxers, ABCA1 and CLA 1, and their extracellular lipid acceptor, apoA‐1, are recruited to the inclusion of C. trachomatis‐infected cells. Furthermore, the apoA‐1 that accumulates within the inclusion colocalizes with pools of phosphatidylcholine. Knockdown of ABCA1, which mediates the cellular efflux of cholesterol and phospholipids to initiate the formation of HDL in the serum, prevents the growth of C. trachomatis in infected HeLa cells. In addition, drugs that inhibit the lipid transport activities of ABCA1 and CLA 1 also inhibit the recruitment of phospholipids to the inclusion and prevent chlamydial growth.These results strongly suggest that C. trachomatis co‐opts the host cell lipid transport system involved in the formation of HDL to acquire lipids, such as phosphatidylcholine, that are necessary for growth.     

Chlamydia trachomatis GlgA Is Secreted into Host Cell Cytoplasm

Glycogen has been localized both inside and outside Chlamydia trachomatis organisms. We now report that C. trachomatis glycogen synthase (GlgA) was detected in both chlamydial organism-associated and -free forms. The organism-free GlgA molecules were localized both in the lumen of chlamydial inclusions and in the cytosol of host cells. The cytosolic GlgA displayed a distribution pattern similar to that of a known C. trachomatis-secreted protease, CPAF. The detection of GlgA was specific since the anti-GlgA antibody labeling was only removed by preabsorption with GlgA but not CPAF fusion proteins. GlgA was detectable at 12h and its localization into host cell cytosol only became apparent at 24h after infection. The cytosolic localization of GlgA was conserved among all C. trachomatis serovars. However, the significance of the GlgA secretion into host cell cytoplasm remains unclear since, while expression of chlamydial GlgA in HeLa cells increased glycogen stores, it did not affect a subsequent infection with C. trachomatis. Similar to several other C. trachomatis-secreted proteins, GlgA is immunogenic in women urogenitally infected with C. trachomatis, suggesting that GlgA is expressed and may be secreted into host cell cytosol during C. trachomatis infection in humans. These findings have provided important information for further understanding C. trachomatis pathogenic mechanisms.     

Effects of Temperature and Dietary Lipids on Phospholipid Fatty Acids and Membrane Fluidity in Steinernema carpocapsae

The phospholipid composition of Steinernema carpocapsae was studied in relation to diet and culture temperature. When reared at 18 and 27.5 C on Galleria mellonella or on an artificial diet supplemented with lard, linseed oil, or fish oil as lipid sources, nematode phospholipids contained an abundance of 20-carbon polyunsaturated fatty acids, with eicosapentaenoic acid (20:5(n - 3)) predominant, regardless of the fatty acid composition of the diet. Because the level of linolenic acid (18:3(n - 3)) in nematode phospholipids was very low and because eicosapentaenoic acid was present even when its precursor (linolenic acid) was undetectable in the diet, S. carpocapsae likely produces n - 3 polyunsaturated fatty acids by de novo biosynthesis, a pathway seldom reported in eukaryotic animals. Reduction of growth temperature from 25 to 18 C increased the proportion of 20:5(n - 3) but not other polyunsaturated fatty acids. A fluorescence polarization technique revealed that vesicles produced from phospholipids of nematodes reared at 18 C were less ordered than those from nematodes reared at 27.5 C, especially in the outermost region of the bilayer. Dietary fish oil increased fluidity in the outermost region but increased rigidity in deeper regions. Therefore, S. carpocapsae appears to modify its membrane physical state in response to temperature, and eicosapentaenoic acid may be involved in this response. The results also indicate that nematode membrane physical state can be modified dietarily, possibly to the benefit of host-finding or survival of S. carpocapsae at low temperatures.     

Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms

Over one hundred different phospholipid molecular species are known to be present in mammalian cells and tissues. Fatty acid remodeling systems for phospholipids including acyl-CoA:lysophospholipid acyltransferases, CoA-dependent and CoA-independent transacylation systems, are involved in the biosynthesis of these molecular species. Acyl-CoA:lysophospholipid acyltransferase system is involved in the synthesis of phospholipid molecular species containing sn-1 saturated and sn-2 unsaturated fatty acids. The CoA-dependent transacylation system catalyzes the transfer of fatty acids esterified in phospholipids to lysophospholipids in the presence of CoA without the generation of free fatty acids. The CoA-dependent transacylation reaction in the rat liver exhibits strict fatty acid specificity, i.e., three types of fatty acids (20:4, 18:2 and 18:0) are transferred. On the other hand, CoA-independent transacylase catalyzes the transfer of C20 and C22 polyunsaturated fatty acids from diacyl phospholipids to various lysophospholipids, especially ether-containing lysophospholipids, in the absence of any cofactors. CoA-independent transacylase is assumed to be involved in the accumulation of PUFA in ether-containing phospholipids. These enzymes are involved in not only the remodeling of fatty acids, but also the synthesis and degradation of some bioactive lipids and their precursors. In this review, recent progresses in acyltransferase research including the identification of the enzyme’s genes are described.     

Lipids of Daucus carota cell suspension cultures

In carrot cell suspension cultures greater amounts of phospholipid were detected than in carrot root material, but the major phospholipid classes were the same in both materials, and their fatty acid composition was very similar. In contrast to the cultured cells, no significant amounts of free fatty acids and monoglycerides, as well as diglycerides, could be detected in the carrot root. The fatty acid composition of the major lipid classes from cell cultures is reported for the first time in this report. The degree of unsaturation was higher in triglycerides and phospholipids than in free fatty acids. In a study of phospholipid biosynthesis, [³H]-glycerol was shown to be incorporated into 4-phospholipids (PE, PC, PG, PS⁷) to different extents. The highest specific activity was observed in PC and PG. Five molecular species were isolated from each of the 4 phospholipids and analyzed by GC-MS and LSC.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl₂ from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which increased in phosphatidylethanolamine content from 39% (low Na⁺) to 48% (high Na⁺) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca²⁺-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na⁺-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca²⁺ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

植物多糖抗肿瘤作用与机理研究——Ⅲ、茯苓多糖(PPS)和刺五加多糖(ASPS)对S180细胞膜脂肪酸组成的影响

我们先前的研究表明,植物多糖抑制体外培养的小鼠肉瘤S180细胞增殖并使细胞膜磷脂含量减少,同时抑制膜磷脂酰肌醇转换。为进一步探讨植物多糖与膜磷脂的关系,本文采用毛细管柱气相色谱法分析了茯苓多糖(PPS)、刺五加多糖(ASPS)与S180细胞一同温育24h后,细胞膜磷脂和中性脂的脂肪酸组成变化,发现中性脂的脂肪酸组成和不饱和性不受影响,磷脂的脂肪酸组成发生明显改变,花生四烯酸(C(20:4))和豆蔻酸(C(14:0))降低(P<0.05或P<0.01),与用作阳性药物对照的氨甲喋呤作用相似。本文对膜磷脂脂肪酸组成变化的意义结合先前的实验结果进行了讨论,认为在PPS、ASPS的抗肿瘤机理中,细胞膜磷脂生化特性的改变是重要环节。     

Peculiarities of the phospholipid and fatty acid composition of erythrocyte plasma membranes of the black sea fish

The phospholipid and the fatty acid composition of the main phospholipid families of erythrocyte plasma membranes was studied in two species of cartilaginous fish: the common thrasher (Raja clavata L.) and the common stingray (Dasyatis pastinaca) and in three bony fish species: the scorpion fish (Scorpaena porcus L.), the smarida (Spicara flexuosa Raf.), and the horse mackerel (Trachurus mediterraneus ponticus Aleev). It was shown that in the studied fish, 70.0–80.0% of all membrane phospholipids were composed of phosphatidylcholine and phosphatidylethanolamine. Phosphatidylserine, monophosphoinositide, and sphingomyelin were minor components whose content in the erythrocyte membrane fluctuated from 3.0% to 13.0%. The fatty acid phospholipids composition was represented by a large specter of acids. From saturated acids, basic for plasma membranes are palmitic (C16: 0) and stearic (C18: 0) acids. From unsaturated acids, the larger part belongs to mono-, tetra-, penta-, and hexaenoic acids in fish phospholipids. The calculation of the double bond index and of the unsaturation coefficient showed difference in the deformation ability of erythrocyte membranes of the studied fish.     

Lipid molecular species composition in developing soybean cotyledons

The fatty acid composition of triglyceride and phospholipids in developing soybean cotyledons (Glycine max L., var. “Harosoy 63”) was analyzed at several stages of growth between 30 and 70 days after flowering. Changes observed in fatty acid composition within each lipid class were related to the levels of lipid molecular species present in the oil. Thirteen molecular species of triglyceride were identified in developing cotyledons, however three of these groups: trilinolenic, dilinolenic-monolinoleic, and linolenic-linoleic-oleic triglycerides, were not found in the mature seed. In immature cotyledons, trioleic and trilinoleic triglycerides accounted for 50% of the structures found; the level of these molecules decreased to 24.9% in the mature seed. The dilinoleic-monolinolenic triglycerides increased from 0.4 to 23.4% during cotyledon development. Changes in triglyceride composition were compared to the levels of molecular species for each phospholipid class. Dilinoleic and monosaturated monolinoleic phospholipid species were dominant in all phospholipid classes throughout development.     

Phospholipids and protein adaptation of Pseudomonas sp. to the xenoestrogen tributyltin chloride (TBT)

A tributyltin (TBT)-resistant strain of Pseudomonas sp. isolated from an overworked car filter was tested for its adaptation to TBT. The isolate was checked for organotin degradation ability, as well as membrane lipid and cellular protein composition in the presence of TBT. The phospholipid profiles of bacteria, grown with and without increased amounts of TBT, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. The strain reacted to the biocide by changing the composition of its phospholipids. TBT induced a twofold decline in the amounts of many molecular species of phosphatidylglycerol and an increase in the levels of phosphatidic acid (by 58 %) and phosphatidylethanolamine (by 70 %). An increase in the degree of saturation of phospholipid fatty acids of TBT exposed Pseudomonas sp. was observed. These changes in the phospholipid composition and concentration reflect the mechanisms which support optimal lipid ordering in the presence of toxic xenobiotic. In the presence of TBT the abundances of 16 proteins, including TonB-dependent receptors, porins and peroxidases were modified, which could indicate a contribution of some enzymes to TBT resistance.     

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components

Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells’ antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.     

Temperature acclimation and phospholipid phase transition in hypothalamic membrane phospholipids of garden lizard, Calotes versicolor

Garden lizards, Calotes versicolor, were acclimated to three different temperatures, i.e., 16°C, 26°C and 36°C for a period of 30 days in ‘walk-in-environmental chambers’. The phospholipid profile and fatty acid pattern were analysed in the hypothalamus and brain of the acclimated animals. Hypothalamic and brain membrane phospholipids were prepared and their phase-transition temperatures were recorded using differential scanning calorimetry. Acclimation temperature, phospholipid composition, fatty acids of these phospholipids and the physical state and fluidity of the specific model membranes of hypothalamus (and brain) are intimately inter-related. Evidence is presented for the first time to show a possible correlation between acclimation temperature and phase-transition temperature of hypothalamic phospholipid membrane. A direct physico-chemical basis is suggested for the thermoregulatory process of hypothalamus leading to a better understanding of our knowledge on the origin of thermoregulation.     

Phospholipid metabolism in V79-R membranes composed of phospholipid molecular species containing trans-monoenoic fatty acids

The interrelationship between the inhibition of cell growth and changes in phospholipid molecular species was studied in the presence of elaidic, trans-11-eicosenoic, or brassidic acids in Chinese hamster V79-R cells. The addition of trans-monoenoic fatty acids to the medium inhibited cell growth and caused an increase in the total cellular content of phospholipids. However, there was no difference in the polar head group composition of these phospholipids among all the cells supplemented with trans-monoenoic fatty acids. Exogenous trans-monoenoic fatty acids were incorporated into cellular phospholipids to form novel phospholipid molecular species. Phospholipid synthesizing enzyme activities bound to the membranes composed of phospholipid molecular species of trans-monoenoic fatty acids were determined. Cholinephosphotransferase [EC 2.7.8.2] and ethanolaminephosphotransferase [EC 2.7.8.1] activities were decreased by trans-11-eicosenoic acid, but not changed by elaidic acid. Glycerophosphate acyltransferase [EC 2.3.1.15] activity was increased by elaidic acid and decreased by trans-11-eicosenoic acid. Cholinephosphate cytidylyltransferase [EC 2.7.7.15] activity was not changed by trans-monoenoic fatty acids.     

The fatty acid composition of phospholipids and absorption spectra of lipid extracts from lamprey,frog, and rat erythrocytes

The work studies the content and fatty acid composition of phospholipids as well as the absorption spectra of lipid extracts from red blood cells of poikilothermal and homoiothermal animals at different evolutionary levels. The objects of study include two poikilothermal species, the river lamprey (Lampetra fluviatilis) that uses oxygen dissolved in water, and the common frog (Rana temporaria) that consumes oxygen both from water and from air. A homoithermal animal is the white rat (Rattus rattus) that inhabits the terrestrial-aerial environment. The animals are studied in winter and spring. The phospholipid content in lamprey blood plasma is found to be twice higher than that in its erythrocytes. In the frog and the rat, the ratio is reverse. Determination of the fatty acid lipid composition of red blood cell phospholipids suggests that membranes in the lamprey are denser than in the frog. As for the fatty acids in the erythrocyte fraction of rat blood, they appear to be less diverse, with a double prevalence of saturated acids over unsaturated ones and devoid of long chain (C22) ω3 fatty acids. All of this results in a lower degree of unsaturation and a denser packing of fatty acids in the membrane structures of rat erythrocytes. The mechanism of reversible binding of O₂ molecules to hemoglobin in erythrocytes is discussed. Presumably, the mechanism of interaction between molecules of O₂ and molecules of water prevents the exchange interaction of electrons of the hemoglobin iron atoms with an oxygen molecule. This is confirmed by our obtained absorption spectra, which show that in the lipid extract almost totally devoid of water the heme isolated from erythrocytes is converted to hemin.     

Schistosoma mansoni does not and cannot oxidise fatty acids,but these are used for biosynthetic purposes instead

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and, in their energy metabolism, strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [¹⁴C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidise fatty acids, as no ¹⁴CO₂ production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for Schistosoma japonicum and all other schistosome species that have been sequenced. Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolised by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicate that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium.     

Incorporation of ω6 polyunsaturated fatty acids into phospholipids of the crab Carcinus maenas

The incorporation in vivo of ¹⁴C-18:2 ω6 and ³H-20:4 ω6 fatty acids in phospholipids isolated from gills, hepatopancreas and hemolymph of the crab Carcinus maenas was analysed. PC was the most heavily labelled phospholipid from these ω6-unsaturated fatty acids and appeared to play an important part in the phospholipids metabolism in Crustaceans. The pathway of fatty acids synthesis in phospholipids of C. maenas seems to be similar to those described for mammals. It is at the level of tissue Pl of C. maenas that the renewal of the 20:4 ω6 fatty acid is the most important. It is suggested that the rapid reorganization of phospholipid molecular species composition in the crab is checked by deacylation—reacylation cycle.     

Synthesis of molecular species of phosphatidylcholine and phosphatidylethanolamine by germinating soya bean

The composition and metabolism of phosphatidylcholines and phosphatidylethanomines of germinating soya bean Glycine max have been examined. Both phospholipids have a very similar fatty acid composition and distribution, with saturated acids located at the 1- position. The fatty acid composition and relative amounts of individual molecular species of the two phospholipids were also very similar. The relative amounts of the species were in the order tetraenoic pentaenoic trienoic = dienoic = monoenoic. In contrast, the labelling of the molecular species from choline Me[¹⁴C] or ethanolamine [2-¹⁴C] showed considerable differences. Phosphatidylethanolamine-[¹⁴C] showed 58% label in trienoic, 17% in tetraenoic, 18% in pentaneoic and 5% in dienoic species 48 hr after germination. The equivalent figures for phosphatidylcholine-[¹⁴C] were 37, 34, 13 and 15% respectively. An increase in labelling of the more unsaturated species was seen with time.