Trypanosoma brucei: inhibition of acetyl-CoA carboxylase by haloxyfop

Trypanosoma brucei, a eukaryotic pathogen that causes African sleeping sickness in humans and nagana in cattle, depends on the enzyme acetyl-CoA carboxylase (ACC) for full virulence in mice. ACC produces malonyl-CoA, the two carbon donor for fatty acid synthesis. We assessed the effect of haloxyfop, an aryloxyphenoxypropionate herbicide inhibitor of plastid ACCs in many plants as well as Toxoplasma gondii, on T. brucei ACC activity and growth in culture. Haloxyfop inhibited TbACC in cell lysate (EC(50) 67 μM), despite the presence of an amino acid motif typically associated with resistance. Haloxyfop also reduced growth of bloodstream and procyclic form parasites (EC(50) of 0.8 and 1.2 mM). However, the effect on growth was likely due to off-target effects because haloxyfop treatment had no effect on fatty acid elongation or incorporation into complex lipids in vivo.     

Respiration of bloodstream forms of the parasite Trypanosoma brucei brucei is dependent on a plant-like alternative oxidase

CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants. The parasites contain 206 ng of CoQ9 mg protein-1 which co-sediments with respiratory activity. The redox state of this CoQ responds in a manner consistent with respiratory function: 60% being in the reduced form when substrate is available and the oxidase is blocked; 13% being in the reduced form when the oxidase is functioning and there is no substrate. The addition of CoQ to aceton-extracted cells stimulates salicylhydroxamic acid-sensitive respiration by 56%. After inhibition of respiration by digitonin-mediated dispersal of the electron transport components, liposomes restore 40% of respiratory activity while liposomes containing CoQ restore 66% of this activity. A less hydrophobic analogue, reduced decyl CoQ, serves as a direct substrate for the trypanosome oxidase supporting full salicylhydroxamic acid-sensitive respiration. After digitonin disruption of electron transport, the nonreduced form of this synthetic substrate can reestablish the chain by accepting electrons from dispersed sn-glycerol-3-phosphate dehydrogenase and transferring them to the dispersed oxidase. Similarities between the alternative oxidase of plants and the oxidase of the trypanosome respiratory system include: mitochondrial location, lack of oxidative phosphorylation, linkage of a dehydrogenase and an oxidase by CoQ, lack of sensitivity to a range of mitochondrial inhibitors, and sensitivity to a spectrum of inhibitors which selectively block transfer of electrons from reduced CoQ to the terminal oxidase but do not block electron transfer to the cytochrome bc1 complex of the mammalian cytochrome chain.     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei

Oxidative phosphorylation and substrate level phosphorylation catalyzed by succinyl-CoA synthetase found in the citric acid and the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle contribute to mitochondrial ATP synthesis in procyclic Trypanosoma brucei. The latter pathway is specific for trypanosome but also found in hydrogenosomes. In organello ATP production was studied in wild-type and in RNA interference cell lines ablated for key enzymes of each of the three pathways. The following results were obtained: 1) ATP production in the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle was directly demonstrated. 2) Succinate dehydrogenase appears to be the only entry point for electrons of mitochondrial substrates into the respiratory chain; however, its activity could be ablated without causing a growth phenotype. 3) Growth of procyclic T. brucei was not affected by the absence of either a functional citric acid or the acetate:succinate CoA transferase/succinyl-CoA synthetase cycle. However, interruption of both pathways in the same cell line resulted in a growth arrest. In summary, these results show that oxygen-independent substrate level phosphorylation either linked to the citric acid cycle or tied into acetate production is essential for growth of procyclic T. brucei, a situation that may reflect an adaptation to the partially hypoxic conditions in the insect host.     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

The ACC1 gene,encoding acetyl-CoA carboxylase,is essential for growth in Ustilago maydis

Acetyl-CoA carboxylase [ACCase; acetylCoA: carbon dioxide ligase (ADP forming), EC 6.4.1.2] catalyses the ATP-dependent carboxylation of acetylCoA to form malonyl-CoA. We have amplified a fragment of the biotin carboxylase (BC) domain of the Ustilago maydis acetyl-CoA carboxylase (ACC1) gene from genomic DNA and used this amplified DNA fragment as a probe to recover the complete gene from a λEMBL3 genomic library. The ACC1 gene has a reading frame of 6555 nucleotides, which is interrupted by a single intron of 80 bb in length. The gene encodes a protein containing 2185 amino acids, with a calculated M_r of 242 530; this is in good agreement with the size of ACCases from other sources. Further identification was based on the position of putative binding sites for acetyl-CoA, ATP, biotin and carboxybiotin found in other ACCases. A single ACC1 allele was disrupted in a diploid wild-type strain. After sporulation of diploid disruptants, no haploid progeny containing a disrupted acc1 allele were recovered, even though an exogenous source of fatty acids was provided. The data indicate that, in U. maydis, ACCase is required for essential cellular processes other than de novo fatty acid biosynthesis.     

Trypanosoma brucei brucei: endocytic recycling is important for mouse infectivity

Endocytosis in the African trypanosome, Trypanosoma brucei, is intimately involved in maintaining homeostasis of the cell surface proteome, morphology of the flagellar pocket and has recently been demonstrated as a bona fide drug target. RNAi-mediated knockdown of many factors required for endocytic transport, including several small GTPases, the major coat protein clathrin and a clathrin-associated receptor, epsinR, results in rapid cell death in vitro. Rapid loss of viability in vitro precludes meaningful investigation by RNAi of the roles of trypanosome endocytosis in vivo. Here we have sought to address this issue using strategies designed to produce milder effects on the endocytic system than complete functional ablation. We created a trypanosome clathrin heavy chain hemizygote and several lines expressing mutant forms of Rab5 and Rab11, described previously. All are viable in in vitro culture, with negligible impact to proliferative rates or cell cycle. Clathrin hemizygotes express clathrin heavy chain at ∼50% of wild type levels, but despite this demonstrate no defect to growth in mice, while none of the Rab5 mutants affected proliferation in vivo, despite clear evidence for effects on endocytosis. By contrast we find that expressing a dominantly active Rab11 mutant led to compromised growth in mice. These data indicate that trypanosomes likely tolerate the effects of partly decreased clathrin expression and alterations in early endocytosis, but are more sensitive to alterations in the recycling arm of the pathway.     

Clathrin-mediated endocytosis is essential in Trypanosoma brucei

In Trypanosoma brucei, the plasma membrane is dominated by glycosylphosphatidylinositol (GPI)-anchored proteins. Endocytic activity correlates with expression levels of the clathrin heavy chain TbCLH, and additional evidence suggests that rapid endocytosis may play a role in evasion of the immune response. TbCLH is present on both endocytic vesicles and post-Golgi elements, suggesting a similar range of functions in trypanosomes to higher eukaryotes. We have assessed the role of TbCLH using RNA interference (RNAi). Suppression of TbCLH expression results in rapid lethality in the bloodstream stage, the form most active for endocytosis. The flagellar pocket, the site of both endocytosis and exocytosis, becomes massively enlarged, suggesting that membrane delivery is unaffected but removal is blocked. Endocytosis in TbCLHRNAi cells is essentially undetectable, suggesting that clathrin-mediated mechanisms are the major route for endocytosis in T.brucei and hence that GPI-anchored proteins are endocytosed by clathrin-dependent pathways in trypanosomes. In contrast, a massive internal accumulation of vesicles and significant alterations to trafficking of a lysosomal protein were observed in the procyclic stage, indicating developmental variation in clathrin function in trypanosomes.     

TbMP81 is required for RNA editing in Trypanosoma brucei

Most mitochondrial mRNAs are edited in Trypano soma brucei by a series of steps that are catalyzed by a multienzyme complex that is in its initial stages of characterization. RNA interference (RNAi)-mediated repression of the expression of TbMP81, a zinc finger protein component of the complex, inhibited growth of bloodstream and insect forms, and blocked in vivo RNA editing. This repression preferentially inhibited insertion editing compared with deletion editing in vitro. It resulted in reduced specific endoribonucleolytic cleavage and a greater reduction of U addition and associated RNA ligation activities than U removal and associated RNA ligation activities. The repressed cells retained 20S editing complexes with several demonstrable proteins and adenylatable TbMP52 RNA ligase, but adenlyatable TbMP48 was not detected. Elimination of TbMP48 by RNAi repression did not inhibit cell growth or in vivo editing in either bloodstream or procyclic forms. These results indicate that TbMP81 is required for RNA editing and suggest that the editing complex is functionally partitioned.     

Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei

In Trypanosoma brucei, the products of two genes, ESAG 6 and ESAG 7, located upstream of the variant surface glycoprotein gene in a polycistronic expression site form a glycosylphosphatidylinositol- anchored transferrin-binding protein (TFBP) complex. It is shown by gel filtration and membrane-binding experiments that the TFBP complex is heterodimeric and binds one molecule of transferrin with high affinity (2,300 binding sites per cell; KD = 2.1 nM for the dominant expression site from T. brucei strain 427 and KD = 131 nM for ES1.3A of the EATRO 1125 stock). The ternary transferrin-TFBP complexes with iron-loaded or iron-free ligand are stable between pH 5 and 8. Cellular transferrin uptake can be inhibited by 90% with Fab fragments from anti-TFBP antibodies. After uptake, the TFBP complex and its ligand are routed to lysosomes where transferrin is proteolytically degraded. While the degradation products are released from the cells, iron remains cell associated and the TFBP complex is probably recycled to the membrane of the flagellar pocket, the only site for exo- and endocytosis in this organism. It is concluded that the TFBP complex serves as the receptor for the uptake of transferrin in T. brucei by a mechanism distinct from that in mammalian cells.     

CYP51 from Trypanosoma brucei is obtusifoliol-specific

New isoforms of CYP51 (sterol 14alpha-demethylase), an essential enzyme in sterol biosynthesis and primary target of azole antimycotic drugs, are found in pathogenic protists, Trypanosoma brucei(TB), T. vivax, T. cruzi, and Leishmania major. The sequences share approximately 80% amino acid identity and are approximately 25% identical to sterol 14alpha-demethylases from other biological kingdoms. Differences of residues conserved throughout the rest of the CYP51 family that align with the BC-loop and helices F and G of CYP51 from Mycobacterium tuberculosis (MT)) imply possible alterations in the topology of the active site cavity of the protozoan enzymes. CYP51 and cytochrome P450 reductase (CPR) from TB were cloned, expressed in Escherichia coli, and purified. The P450 has normal spectral features (including absolute absorbance, carbon monoxide, and ligand binding spectra), is efficiently reduced by TB and rat CPR but demonstrates altered specificity in comparison with human CYP51 toward three tested azole inhibitors, and contrary to the human, Candida albicans, and MT isoforms, reveals profound substrate preference toward obtusifoliol (turnover 5.6 min(-1)). It weakly interacts with the other known CYP51 substrates; slow lanosterol conversion predominantly produces the 14alpha-carboxyaldehyde intermediate. Although obtusifoliol specificity is typical for plant isoforms of CYP51, the set of sterol biosynthetic enzymes in the protozoan genomes together with available information about sterol composition of kinetoplastid cells suggest that the substrate preference of TBCYP51 may reflect a novel sterol biosynthetic pathway in Trypanosomatidae.     

Measure of molecular diversity within the Trypanosoma brucei subspecies Trypanosoma brucei brucei and Trypanosoma brucei gambiense as revealed by genotypic characterization

We have evaluated whether sequence polymorphisms in the rRNA intergenic spacer region can be used to study the relatedness of two subspecies of Trypanosoma brucei. Thirteen T. brucei isolates made up of 6 T. b. brucei and 7 T. b. gambiense were analyzed using restriction fragment length polymorphism (RFLP). By PCR-based restriction mapping of the ITS1-5.8S-ITS2 ribosomal repeat unit, we found a fingerprint pattern that separately identifies each of the two subspecies analyzed, with unique restriction fragments observed in all but 1 of the T. b. gambiense "human" isolates. Interestingly, the restriction profile for a virulent group 2 T. b. gambiense human isolate revealed an unusual RFLP pattern different from the profile of other human isolates. Sequencing data from four representatives of each of the two subspecies indicated that the intergenic spacer region had a conserved ITS-1 and a variable 5.8S with unique transversions, insertions, or deletions. The ITS-2 regions contained a single repeated element at similar positions in all isolates examined, but not in 2 of the human isolates. A unique 4-bp [C(3)A] sequence was found within the 5.8S region of human T. b. gambiense isolates. Phylogenetic analysis of the data suggests that their common ancestor was a nonhuman animal pathogen and that human pathogenicity might have evolved secondarily. Our data show that cryptic species within the T. brucei group can be distinguished by differences in the PCR-RFLP profile of the rDNA repeat.     

The de novo synthesis of GDP-fucose is essential for flagellar adhesion and cell growth in Trypanosoma brucei

The protozoan parasite Trypanosoma brucei causes human African sleeping sickness in sub-Saharan Africa. The parasite makes several essential glycoproteins, which has led to the investigation of the sugar nucleotides and glycosyltransferases required to synthesize these structures. Fucose is a common sugar in glycoconjugates from many organisms; however, the sugar nucleotide donor GDP-fucose was only recently detected in T. brucei, and the importance of fucose metabolism in this organism is not known. In this paper, we identified the genes encoding functional GDP-fucose biosynthesis enzymes in T. brucei and created conditional null mutants of TbGMD, the gene encoding the first enzyme in the pathway from GDP-mannose to GDP-fucose, in both bloodstream form and procyclic form parasites. Under nonpermissive conditions, both life cycle forms of the parasite became depleted in GDP-fucose and suffered growth arrest, demonstrating that fucose metabolism is essential to both life cycle stages. In procyclic form parasites, flagellar detachment from the cell body was also observed under nonpermissive conditions, suggesting that fucose plays a significant role in flagellar adhesion. Fluorescence microscopy of epitope-tagged TbGMD revealed that this enzyme is localized in glycosomes, despite the absence of PTS-1 or PTS-2 target sequences.     

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.     

Mouse ornithine decarboxylase is stable in Trypanosoma brucei.

The cDNA encoding mouse ornithine decarboxylase (ODC) was incorporated into a transforming vector pTSA-NEO2 carrying a procyclic acidic repetitive protein promoter and a neomycin phosphotransferase gene. The plasmid thus constructed, pMOD300, was introduced into the procyclic forms of Trypanosoma brucei via electroporation, and the transformants, selected under G418, expressed an ODC activity 100 times above the background level. Contrary to the commonly observed short half-life of mouse ODC in mammalian cells, however, the mouse ODC activity expressed in T. brucei remained stable for at least 6 h when protein synthesis was inhibited by cycloheximide. Pulse labelings and chase experiments with the irreversible ODC inhibitor [3,4-3H]difluoromethylornithine followed by gel electrophoresis, or with L-[35S] methionine followed by immunoprecipitation and gel electrophoresis indicated that the stable mouse ODC expressed in T. brucei has the same subunit molecular weight as the native enzyme. By an in vitro assay of protein stability in rabbit reticulocyte lysates (Loetscher, P., Pratt, G., and Rechsteiner, M. (1991) J. Biol. Chem. 266, 11213-11220), the native mouse ODC and the enzyme expressed in T. brucei had the same degree of instability. Thus, the mouse ODC expressed in T. brucei is probably identical to the native mouse ODC. Its remarkable stability in T. brucei must be due to the absence in trypanosomes of the proteolytic machinery present in mammalian cells responsible for rapid degradation of mouse ODC.     

Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei’s survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC₅₀) of PCF was 1.0 ± 0.2 μM for Isoxyl and 5 ± 2 μM for 10-TS, whereas BSF appeared more susceptible with EC₅₀ values 0.10 ± 0.03 μM (Isoxyl) and 1.0 ± 0.6 μM (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents’ survival.     

Biosynthesis of trypanothione in Trypanosoma brucei brucei

Trypanothione [T(SH)2], the major redox mediator in pathogenic trypanosomatids, is synthetized stepwise by two distinct enzymes in Crithidia fasciculata, while in Trypanosoma cruzi a single enzyme catalyzes both steps. A full-length reading frame presumed to encode trypanothione synthetase (TryS) was obtained by PCR using DNA of T. brucei as template and primers based on fragments of putative TryS genes. The recombinant protein produced by E. coli Origami (DE3) was purified to homogeneity by chelate and ion exchange chromatography. The enzyme catalyzed both reactions of T(SH)2 biosynthesis. Thus, T(SH)2 synthesis appears to be similar in African (T. brucei) and New World (T. cruzi) trypanosomes but distinct from that of Crithidia.