Phosphatidylinositol synthesis is essential in bloodstream form Trypanosoma brucei

PI (phosphatidylinositol) is a ubiquitous eukaryotic phospholipid which serves as a precursor for messenger molecules and GPI (glycosylphosphatidylinositol) anchors. PI is synthesized either de novo or by head group exchange by a PIS (PI synthase). The synthesis of GPI anchors has previously been validated both genetically and chemically as a drug target in Trypanosoma brucei, the causative parasite of African sleeping sickness. However, nothing is known about the synthesis of PI in this organism. Database mining revealed a putative TbPIS gene in the T. brucei genome and by recombinant expression and characterization it was shown to encode a catalytically active PIS, with a high specificity for myo-inositol. Immunofluorescence revealed that in T. brucei, PIS is found in both the endoplasmic reticulum and Golgi. We created a conditional double knockout of TbPIS in the bloodstream form of T. brucei, which when grown under non-permissive conditions, clearly showed that TbPIS is an essential gene. In vivo labelling of these conditional double knockout cells confirmed this result, showing a decrease in the amount of PI formed by the cells when grown under non-permissive conditions. Furthermore, quantitative and qualitative analysis by GLC-MS and ESI-MS/MS (electrospray ionization MS/MS) respectively showed a significant decrease (70%) in cellular PI, which appears to affect all major PI species equally. A consequence of this fall in PI level is a knock-on reduction in GPI biosynthesis which is essential for the parasite's survival. The results presented here show that PI synthesis is essential for bloodstream form T. brucei, and to our knowledge this is the first report of the dependence on PI synthesis of a protozoan parasite by genetic validation.     

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.     

The de novo and salvage pathways of GDP-mannose biosynthesis are both sufficient for the growth of bloodstream-form Trypanosoma brucei

The sugar nucleotide GDP-mannose is essential for Trypanosoma brucei. Phosphomannose isomerase occupies a key position on the de novo pathway to GDP-mannose from glucose, just before intersection with the salvage pathway from free mannose. We identified the parasite phosphomannose isomerase gene, confirmed that it encodes phosphomannose isomerase activity and localized the endogenous enzyme to the glycosome. We also created a bloodstream-form conditional null mutant of phosphomannose isomerase to assess the relative roles of the de novo and salvage pathways of GDP-mannose biosynthesis. Phosphomannose isomerase was found to be essential for parasite growth. However, supplementation of the medium with low concentrations of mannose, including that found in human plasma, relieved this dependence. Therefore, we do not consider phosphomannose isomerase to be a viable drug target. We further established culture conditions where we can control glucose and mannose concentrations and perform steady-state [U-(13) C]-D-glucose labelling. Analysis of the isotopic sugar composition of the parasites variant surface glycoprotein synthesized in cells incubated in 5 mM [U-(13) C]-D-glucose in the presence and absence of unlabelled mannose showed that, under physiological conditions, about 80% of GDP-mannose synthesis comes from the de novo pathway and 20% from the salvage pathway.     

Role of exogenous inositol and phosphatidylinositol in glycosylphosphatidylinositol anchor synthesis of GP49 by Giardia lamblia

Although Giardia lamblia trophozoites are unable to carry out de novo phospholipid synthesis, they can assemble complex glycophospholipids from simple lipids and fatty acids acquired from the host. Previously, we have reported that G. lamblia synthesizes GP49, an invariant surface antigen with a glycosylphosphatidylinositol (GPI) anchor. It is therefore possible that myo-inositol (Ins), phosphatidylinositol (PI) and other GPI precursors are obtained from the dietary products of the human small intestine, where the trophozoites colonize. In this report, we have investigated the role of exogenous Ins and PI on GPI anchor synthesis by G. lamblia. The results demonstrate that [(3)H]Ins and PI internalized by trophozoites, metabolically transformed into GlcN(acyl)-PI and downstream GPI molecules. Further investigations suggest that G. lamblia expresses cytidine monophosphate (CMP)-dependent (Mg(2+)-stimulated) and independent (Mn(2+)-stimulated) inositol headgroup exchange enzymes, which are responsible for exchanging free Ins with cellular PI. We observed that 3-deoxy-3-fluoro-D-myo-inositol (3-F-Ins) and 1-deoxy-1-F-scyllo-Ins (1-F-scyllo-Ins), which are considered potent inhibitors of Mn(2+)-stimulated headgroup exchange enzyme, inhibited the incorporation of [(3)H]Ins into PI and GPI molecules significantly, suggesting that CMP-independent (Mn(2+)-stimulated) exchange enzyme may be important for these reactions. However, 3-F-Ins and 1-F-scyllo-Ins were not effective in blocking the incorporation of exogenously supplied [(3)H]PI into GPI glycolipids. Thus, it can be concluded that G. lamblia can use exogenously supplied [(3)H]PI and [(3)H]Ins to synthesize GPI glycolipids of GP49; while PI is directly incorporated into GPI molecules, free Ins is first converted into PI by headgroup exchange enzymes, and this newly formed PI participates in GPI anchor synthesis.     

Plasmodium falciparum is dependent on de novo myo‐inositol biosynthesis for assembly of GPI glycolipids and infectivity

Intra‐erythrocytic stages of the malaria parasite, Plasmodium falciparum, are thought to be dependent on de novo synthesis of phosphatidylinositol, as red blood cells (RBC) lack the capacity to synthesize this phospholipid. The myo‐inositol headgroup of PI can either be synthesized de novo or scavenged from the RBC. An untargeted metabolite profiling of P. falciparum infected RBC showed that trophozoite and schizont stages accumulate high levels of myo‐inositol‐3‐phosphate, indicating increased de novo biosynthesis of myo‐inositol from glucose 6‐phosphate. Metabolic labelling studies with ¹³C‐U‐glucose in the presence and absence of exogenous inositol confirmed that de novo myo‐inositol synthesis occurs in parallel with myo‐inositol salvage pathways. Unexpectedly, while both endogenous and scavenged myo‐inositol was used to synthesize bulk PI, only de novo‐synthesized myo‐inositol was incorporated into GPI glycolipids. Moreover, gene disruption studies suggested that the INO1 gene, encoding myo‐inositol 3‐phosphate synthase, is essential in asexual parasite stages. Together these findings suggest that P. falciparum asexual stages are critically dependent on de novo myo‐inositol biosynthesis for assembly of a sub‐pool of PI species and GPI biosynthesis. These findings highlight unexpected complexity in phospholipid biosynthesis in P. falciparum and a lack of redundancy in some nutrient salvage versus endogenous biosynthesis pathways.     

Uptake and metabolism of myo-inositol by L1210 leukaemia cells.

The initial rate of uptake of [3H]myo-inositol by L1210 murine leukaemia cells is directly proportional to the extracellular concentration and unaffected by several analogues of myo-inositol even at millimolar concentrations. Scyllitol, a geometric isomer of myo-inositol, partially inhibited the uptake of myo-inositol (40% at 0.1 mM). A portion of the uptake of myo-inositol was not inhibited even at 5 mM-scyllitol. At steady-state the intracellular concentration of [3H]myo-inositol is directly proportional to the extracellular concentration. Addition of myo-inositol to medium does not enhance the growth of L1210 cells; these cells can maintain an extracellular concentration of 20 microM-myo-inositol even when grown in myo-inositol-free medium. Synthesis of myo-inositol from glucose by L1210 cells was demonstrated by use of [13C]glucose and m.s. L1210 cells maintain myo-inositol pools by a combination of synthesis de novo and uptake of exogenous myo-inositol by either passive diffusion or a low affinity carrier.     

Parasite-specific inhibition of the glycosylphosphatidylinositol biosynthetic pathway by stereoisomeric substrate analogues

The natural substrate for the first alpha-D-mannosyltransferase of glycosylphosphatidylinositol biosynthesis in the protozoan parasite Trypanosoma brucei is D-GlcNalpha1-6-D-myo-inositol-1-P-sn-1, 2-diacylglycerol. Here we show that a diastereoisomer, D-GlcNalpha1-6-L-myo-inositol-1-P-sn-1,2-diacylglycerol, is an inhibitor of this enzyme in a trypanosomal cell-free system. Tests with other L-myo-inositol-containing compounds revealed that L-myo-inositol-1-phosphate is the principal inhibitory component and that methylation of the 2-OH group of the L-myo-inositol residue abolishes any inhibition. Comparisons between the natural substrate and the inhibitors suggested that the inhibitors bind to the first alpha-D-mannosyltransferase by means of charge interactions with the 1-phosphate group and/or hydrogen bonds involving the 3-, 4-, and 5-OH groups of the L-myo-inositol residue, which are predicted to occupy orientations identical to those of the 1-phosphate and 5-, 4-, and 3-OH groups, respectively, of the D-myo-inositol residue of the natural substrate. However, additional experiments indicated that the 4-OH group of the D-myo-inositol residue is unlikely to be involved in substrate recognition. None of the L-myo-inositol-containing compounds that inhibited glycosylphosphatidylinositol (GPI) biosynthesis in a parasite cell-free system had any effect on GPI biosynthesis in a comparable human (HeLa) cell-free system, suggesting that other related parasite-specific inhibitors of this essential pathway might be developed.     

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.     

Sphingolipid metabolism is a crucial determinant of cellular fate in nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.     

A glycosylphosphatidylinositol protein anchor from procyclic stage Trypanosoma brucei: lipid structure and biosynthesis.

Cells of the insect (procyclic) stage of the life cycle of the African trypanosome, Trypanosoma brucei, express an abundant stage-specific glycosylated phosphatidylinositol (GPI) anchored glycoprotein, the procyclic acidic repetitive protein (PARP). The anchor is insensitive to the action of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting that it contains an acyl-inositol. We have recently described the structure of a PI-PLC resistant glycosylphosphatidylinositol, PP1, which is specific to the procyclic stage, and have presented preliminary evidence that the phosphatidylinositol portion of the protein-linked GPI on PARP has a similar structure. In this paper we show, by metabolic labelling with [3H]fatty acids, that the PARP anchor contains palmitate esterified to inositol, and stearate at sn-1, in a monoacylglycerol moiety, a structure identical to PP1. Using pulse-chase labelling, we show that both fatty acids are incorporated into the GPI anchor from a large pool of metabolic precursors, rather than directly from acyl-CoA. We also demonstrate that the addition of the GPI anchor moiety to PARP is dependent on de novo protein synthesis, excluding the possibility that incorporation of fatty acids into PARP can occur by a remodelling of pre-existing GPI anchors. Finally we show that the phosphatidylinositol (PI) species that are utilized for GPI biosynthesis are a subpopulation of the cellular PI molecular species. We propose that these observations may be of general validity since several other eukaryotic membrane proteins (e.g. human erythrocyte acetylcholine esterase and decay accelerating factor) have been reported to contain palmitoylated inositol residues.     

Repression of polymerase I-mediated gene expression at Trypanosoma brucei telomeres

The African trypanosome, Trypanosoma brucei, is a flagellated pathogenic protozoan that branched early from the eukaryotic lineage. Unusually, it uses RNA polymerase I (Pol I) for mono-telomeric expression of variant surface glycoprotein (VSG) genes in bloodstream-form cells. Many other subtelomeric VSG genes are reversibly repressed, but no repressive DNA sequence has been identified in any trypanosomatid. Here, we show that artificially seeded de novo telomeres repress Pol I-dependent gene expression in mammalian bloodstream and insect life-cycle stages of T. brucei. In a telomeric VSG expression site, repression spreads further along the chromosome and this effect is specific to the bloodstream stage. We also show that de novo telomere extension is telomerase dependent and that the rate of extension correlates with the expression level of the adjacent gene. Our results show constitutive telomeric repression in T. brucei and indicate that an enhanced, developmental stage-specific repression mechanism controls antigenic variation.     

Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis.

Glycosylphosphatidylinositol (GPI)-anchoring represents a mechanism for attaching proteins to the cell surface that is used among all eukaryotes. A common core structure, EthN-P-Man3-GlcN-PI, is synthesized by sequential transfer of sugars and ethanolamine-P to PI and is highly conserved between organisms. We have screened for natural compounds that inhibit GPI-anchoring in yeast and have identified a terpenoid lactone, YW3548, that specifically blocks the addition of the third mannose to the intermediate structure Man2-GlcN-acyIPI. Consistent with the block in GPI synthesis, YW3548 prevents the incorporation of [3H]myo-inositol into proteins, transport of GPI-anchored proteins to the Golgi and is toxic. The compound inhibits the same step of GPI synthesis in mammalian cells, but has no significant activity in protozoa. These results suggest that despite the conserved core structure, the GPI biosynthetic machinery may be different enough between mammalian and protozoa to represent a target for anti-protozoan chemotherapy.     

Segregation of glycosylphosphatidylinositol biosynthetic reactions in a subcompartment of the endoplasmic reticulum.

Glycosylphosphatidylinositols (GPIs) are synthesized in the endoplasmic reticulum (ER) via the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI). While attempting to establish a mammalian cell-free system for GPI biosynthesis, we found that the assembly of mannosylated GPI species was impaired when purified ER preparations were substituted for unfractionated cell lysates as the enzyme source. To explore this problem we analyzed the distribution of the various GPI biosynthetic reactions in subcellular fractions prepared from homogenates of mammalian cells. The results indicate the following: (i) the initial reaction of GPI assembly, i.e. the transfer of GlcNAc to PI to form GlcNAc-PI, is uniformly distributed in the ER; (ii) the second step of the pathway, i.e. de-N-acetylation of GlcNAc-PI to yield GlcN-PI, is largely confined to a subcompartment of the ER that appears to be associated with mitochondria; (iii) the mitochondria-associated ER subcompartment is enriched in enzymatic activities involved in the conversion of GlcN-PI to H5 (a singly mannosylated GPI structure containing one phosphoethanolamine side chain; and (iv) the mitochondria-associated ER subcompartment, unlike bulk ER, is capable of the de novo synthesis of H5 from UDP-GlcNAc and PI. The confinement of these GPI biosynthetic reactions to a domain of the ER provides another example of the compositional and functional heterogeneity of the ER. The implications of these findings for GPI assembly are discussed.     

Localization of myo-inositol phosphate synthase (GmMIPS-1) during the early stages of soybean seed development

As a precursor to a large variety of compounds, myo-inositol is a central molecule required for cell metabolism and plant growth. The de novo synthesis of myo-inositol requires the activity of the enzyme D-myo-inositol-3-phosphate synthase (MIPS). MIPS cDNAs encoding one or more isoforms have been cloned from a number of species, nevertheless, little is known about the regulation of MIPS expression in developing seed. Seed-specific expression of a soybean isoform (GmMIPS-1) has been demonstrated, but tissue-specific localization during embryo development has not been reported. Using immunolocalization techniques, a specialized area of GmMIPS-1 expression was identified in the outer integumentary layer during early soybean seed development. In addition, localization data provided evidence that MIPS was associated with oxalate crystal idioblasts.     

Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

Synthetic analogues of D-GlcNalpha1-6D-myo-inositol-1-HPO(4)-3(sn-1, 2-diacylglycerol) (GlcN-PI), with the 2-position of the inositol residue substituted with an O-octyl ether [D-GlcNalpha1-6D-(2-O-octyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-octyl) PI] or O-hexadecyl ether [D-GlcNalpha1-6D-(2-O-hexadecyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell-free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N-acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell-free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell-free system. GlcN-(2-O-hexadecyl)PI was shown to inhibit the first alpha-mannosyltransferase of the trypanosomal GPI pathway. The N-acetylated derivative GlcNAc-(2-O-octyl)PI is a substrate for the trypanosomal GlcNAc-PI de-N-acetylase and this compound, like GlcN-(2-O-octyl)PI, is processed predominantly to Man(2)GlcN-(2-O-octyl)PI by the T.brucei cell-free system. Both GlcN-(2-O-octyl)PI and GlcNAc(2-O-octyl)PI also inhibit inositol acylation of Man(1-3)GlcN-PI and, consequently, the addition of the ethanolamine phosphate bridge in the T.brucei cell-free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway-specific inhibitors.     

In vitro and ex vivo effects of antidepressants on rat brain membrane-bound phosphatidylinositol synthetase activity

The in vitro and ex vivo effects of antidepressant drugs on membrane-bound phosphatidylinositol (PI) synthetase and PI: myo-inositol exchange enzyme activities were examined. In rat brain subcellular fractions, PI synthetase occurred exclusively in the microsomes. In comparison, the activity of CDP-diglyceride independent PI: myo-inositol exchange enzyme was low (3%). Of the various CDP-diglycerides tested for the activation of PI synthetase, CDP-dipalmitin was the most active. Addition of 1 mM of desipramine, amitriptyline, imipramine, iprindole, clomipramine and mianserin in vitro significantly inhibited (30–60%) PI synthetase activity, whereas the same concentration of zimelidine and fluoxetine had no effect. At low liponucleotide concentrations, PI synthetase activity was significantly enhanced by imipramine (1 mM), whereas the enzyme activity was inhibited at higher liponucleotide concentrations (>0.3 mM). In contrast, imipramine had no effect on the PI: myo-inositol exchange enzyme activity. No significant alteration in the PI synthetase activity was found following either acute (2 h) or chronic (21 d) treatment of rats with imipramine. The above results indicate that the de novo synthesis of PI is inhibited in vitro but not ex vivo by some antidepressant drugs. However, in view of the high concentration of the drugs required, the pharmacological significance of this inhibitory action with respect to their therapeutic effects is doubtful.