Cargo selection in the early secretory pathway of African trypanosomes

Membrane and secretory proteins are synthesized by ribosomes and then enter the endoplasmic reticulum (ER) where they undergo glycosylation and quality control for proper folding. Subsequently, proteins are transported to the Golgi apparatus and then sorted to the plasma membrane or intracellular organelles. Transport vesicles are formed at ER-exit sites (ERES) on the ER with several coat protein complexes. Cargo proteins loaded into the vesicles are selected by specific interactions with cargo receptors and/or adaptors during vesicle formation. p24 family and intracellular lectin ERGIC-53-membrane proteins are the known cargo receptors acting in the early secretory pathway (ER-Golgi). Oligomerization of the cargo receptors have been suggested to play an important role in cargo selection and sorting via posttranslational modifications in fungi and metazoans. On the other hand, the mechanisms involved in the early secretory pathway in protozoa remain unclear. In this review, we focus on Trypanosoma brucei as a representative of protozoan and discuss differences and commonalities in the molecular mechanisms of its early secretory pathway compared with other organisms.     

Assembling the components of the quorum sensing pathway in African trypanosomes

African trypanosomes, parasites that cause human sleeping sickness, undergo a density‐dependent differentiation in the bloodstream of their mammalian hosts. This process is driven by a released parasite‐derived factor that causes parasites to accumulate in G1 and become quiescent. This is accompanied by morphological transformation to ‘stumpy’ forms that are adapted to survival and further development when taken up in the blood meal of tsetse flies, the vector for trypanosomiasis. Although the soluble signal driving differentiation to stumpy forms is unidentified, a recent genome‐wide RNAi screen identified many of the intracellular signalling and effector molecules required for the response to this signal. These resemble components of nutritional starvation and quiescence pathways in other eukaryotes, suggesting that parasite development shares similarities with the adaptive quiescence of organisms such as yeasts and Dictyostelium in response to nutritional starvation and stress. Here, the trypanosome signalling pathway is discussed in the context of these conserved pathways and the possible contributions of opposing ‘slender retainer’ and ‘stumpy inducer’ arms described. As evolutionarily highly divergent eukaryotes, the organisation and conservation of this developmental pathway can provide insight into the developmental cycle of other protozoan parasites, as well as the adaptive and programmed developmental responses of all eukaryotic cells.     

Fatty acid synthesis in African trypanosomes: a solution to the myristate mystery.

The glycosyl phosphatidylinositol anchor of the trypanosome variant surface glycoprotein contains myristate as its sole fatty acid component. Surprisingly, there does not appear to be enough myristate in either the parasite or its host's bloodstream to sustain myristoylation of the enormous quantity of variant surface glycoprotein produced. Here, we discuss how the trypanosome solves its myristate dilemma. The parasite not only efficiently salvages and processes myristate from the bloodstream, but it also makes myristate de novo using a recently discovered specialized fatty acid synthesis system.     

The role of intermediates in mitochondrial fatty acid oxidation.

1. Rat liver mitochondria oxidizing [16-14C]palmitoylcarnitine accumulate saturated long-chain thiester intermediates which may be detected by radio-g.1.c.2. Time-courses of intermediate accumulation display no product-precursor relationships and the end product, measured as [14C]citrate, is produced without a detectable initial lag. 3. A short pulse of [16-14C]palmitoylcarnitine followed by unlabelled palmitoylcarnitine showed that the observed intermediates(at least in the greater part)were not the direct precursors of [14C]citrate. 4. The quantity of saturated intermediates depended on the total accumulated flux of acyl units through the pathway provided that some mitochondrial CoA and unused substrate remained. 5. In the presence of rotenone and carnitine, 2-unsaturated, 3-unsaturated and 3-hydroxy intermediates were formed as well as saturated intermediates...     

An update on antigenic variation in African trypanosomes.

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?     

Phosphatidyl choline fatty acid remodeling in the hepatic cell nuclei

This study was performed to determine whether fatty acids incorporated into liver cell nuclei phosphatidylcholine (PtdCho) could be remodeled in the isolated nuclear. For this reason, rat liver cell nuclei were incubated in vitro with [1-14C]20:4n-6-CoA. PtdCho molecular species with the highest specific activity had an unsaturated fatty acid at sn-1 and sn-2 positions (20:4-20:4>18:2-20:4>18:1-20:4). 16:0-20:4 and 18:0-20:4 PtdChos showed a minor specific activity. When labeled nuclei were reincubated in the absence of labeled substrate with the addition of cytosol, ATP and CoA, the specific activity of 20:4-20:4, 18:2-20:4 and 18:1-20:4 species decreased, while that of 16:0-20:4 and 18:0-20:4 increased. In conclusion, the asymmetric fatty acid distribution of saturated fatty acids at sn-1 position, and unsaturated fatty acids at sn-2 position of nuclear PtdCho molecular species was re-established by an acyl-CoA-dependent remodeling process.     

Biology of African trypanosomes in the tsetse fly

African trypanosomes present several features of interest to cell biologists. These include: a repressible single mitochondrion with a large mass of mitochondrial DNA, the kinetoplast; a special organelle, the glycosome, which houses the enzymes of the glycolytic chain; a surface coat of variable glycoprotein which enables the parasite to evade the mammalian host's immune response; and a unique flagellum-to-host attachment mechanism associated with novel cytoskeletal elements. Trypanosome development during the life cycle involves cyclical activation and repression of genes controlling these activities. Understanding the complexity of parasite development in the tsetse fly vector is especially challenging but may help to suggest new methods for the control of trypanosomiasis.     

Resolution of the species problem in African trypanosomes

There is a general assumption that eukaryote species are demarcated by morphological or genetic discontinuities. This stems from the idea that species are defined by the ability of individuals to mate and produce viable progeny. At the microscopic level, where organisms often proliferate more by asexual than sexual reproduction, this tidy classification system breaks down and species definition becomes messy and problematic. The dearth of morphological characters to distinguish microbial species has led to the widespread application of molecular methods for identification. As well as providing molecular markers for species identification, gene sequencing has generated the data for accurate estimation of relatedness between different populations of microbes. This has led to recognition of conflicts between current taxonomic designations and phylogenetic placement. In the case of microbial pathogens, the extent to which taxonomy has been driven by utilitarian rather than biological considerations has been made explicit by molecular phylogenetic analysis. These issues are discussed with reference to the taxonomy of the African trypanosomes, where pathogenicity, host range and distribution have been influential in the designation of species and subspecies. Effectively, the taxonomic units recognised are those that are meaningful in terms of human or animal disease. The underlying genetic differences separating the currently recognised trypanosome taxa are not consistent, ranging from genome-wide divergence to presence/absence of a single gene. Nevertheless, if even a minor genetic difference reflects adaptation to a particular parasitic niche, for example, in Trypanosoma brucei rhodesiense, the presence of a single gene conferring the ability to infect humans, then it can prove useful as an identification tag for the taxon occupying that niche. Thus, the species problem can be resolved by bringing together considerations of utility, genetic difference and adaptation.     

A molecular mechanism for eflornithine resistance in African trypanosomes

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.     

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C₄), -octanoyl (C₈), -decanoyl (C₁₀), -dodecanoyl (C₁₂) and -tetradecanoyl (C₁₄)-ACPs display marked changes in backbone HN, C_α and C_β chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.     

The biochemical basis of arsenical-diamidine crossresistance in African trypanosomes.

Resistance to currently used drugs is a serious problem in most fields of antimicrobial chemotherapy. Crossresistance between two of the major classes of drug used in the treatment of African trypanosomiasis, the melaminophenyl arsenicals and diamidines is easily selected in the laboratory. Here, Mike Barrett and Alan Fairlamb outline the mechanism underlying this crossresistance, which appears to arise as a result of alterations in an unusual adenosine transporter involved in the uptake of these drugs.     

New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway

Most, if not all, cytokines activate phosphatidylinositol 3-kinase (PI-3K). Although many cytokine receptors have direct binding sites for the p85 subunit of PI-3K, others, such as the interleukin-3 (IL-3) receptor beta common chain (betac) and the IL-2 receptor beta chain (IL-2Rbeta), lack such sites, leaving the mechanism by which they activate PI-3K unclear. Here, we show that the protooncoprotein Shc, which promotes Ras activation by recruiting the Grb2-Sos complex in response to stimulation of cytokine stimulation, also signals to the PI-3K/Akt pathway. Analysis of Y-->F and "add-back" mutants of betac shows that Y577, the Shc binding site, is the major site required for Gab2 phosphorylation in response to cytokine stimulation. When fused directly to a mutant form of IL-2Rbeta that lacks other cytoplasmic tyrosines, Shc can promote Gab2 tyrosyl phosphorylation. Mutation of the three tyrosyl phosphorylation sites of Shc, which bind Grb2, blocks the ability of the Shc chimera to evoke Gab2 tyrosyl phosphorylation. Overexpression of mutants of Grb2 with inactive SH2 or SH3 domains also blocks cytokine-stimulated Gab2 phosphorylation. The majority of cytokine-stimulated PI-3K activity associates with Gab2, and inducible expression of a Gab2 mutant unable to bind PI-3K markedly impairs IL-3-induced Akt activation and cell growth. Experiments with the chimeric receptors indicate that Shc also signals to the PI-3K/Akt pathway in response to IL-2. Our results suggest that cytokine receptors lacking direct PI-3K binding sites activate Akt via a Shc/Grb2/Gab2/PI-3K pathway, thereby regulating cell survival and/or proliferation.     

Fatty acid remodeling: a novel reaction sequence in the biosynthesis of trypanosome glycosyl phosphatidylinositol membrane anchors.

The trypanosome variant surface glycoprotein (VSG) is anchored to the plasma membrane via a glycosyl phosphatidylinositol (GPI). The GPI is synthesized as a precursor, glycolipid A, that is subsequently linked to the VSG polypeptide. The VSG anchor is unusual, compared with anchors in other cell types, in that its fatty acid moieties are exclusively myristic acid. To investigate the mechanism for myristate specificity we used a cell-free system for GPI biosynthesis. One product of this system, glycolipid A', is indistinguishable from glycolipid A except that its fatty acids are more hydrophobic than myristate. Glycolipid A' is converted to glycolipid A through highly specific fatty acid remodeling reactions involving deacylation and subsequent reacylation with myristate. Therefore, myristoylation occurs in the final phase of trypanosome GPI biosynthesis.     

Fluorine-containing analogues of intermediates in the Shikimate pathway.

The phosphoenolpyruvate analogue (Z)-phosphoenol-3-fluoropyruvate is a substrate for phenylalanine-inhibitable 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase from Escherichia coli. In the presence of excess erythrose 4-phosphate, apparent KM values of 65 and 38 muM were observed for phosphoenol-3-fluoropyruvate and phosphoenolpyruvate, respectively. Because the apparent Vmax for phosphoenol-3-fluoropyruvate is only 1.17% of that for phosphoenolpyruvate, one can study the former as an inhibitor of 3-deoxy-arabino-heptulosonic acid-7-phosphate synthase. Kinetic experiments showed phosphoenol-3-fluoropyruvate to be competitive with respect to phosphoenolpyruvate. Two distinguishable Ki values of 8 and 48 muM were obtained. The product (3S)-3-deoxy--3-fluoro-arabino-heptulosonic acid 7-phosphate was purified, characterized, and shown to act as a substrate for 5-dehydroquinate synthase. 3-Deoxy-3-fluoro-arabino-heptulosonic acid 7-phosphate, in contrast to 3-deoxy-arabino-heptulosonic acid 7-phosphate reacts slowly or not at all with reagents specific for 2-keto-3-deoxy sugars and is relatively resistant to oxidative cleavage by sodium periodate. The expected product of periodate oxidation, 3-fluoro-3-formylpyruvate, cannot be detected. This observation was clarified by studies with model compounds.     

Biogenesis of transport intermediates in the endocytic pathway.

Evidence is accumulating that membrane traffic between organelles can be achieved by different types of intermediates. Small (< 100 nm) and short-lived vesicles mediate transport from the plasma membrane or the trans-Golgi network to endosomes, and formation of these vesicles depends on specific adapter complexes. In contrast, transport from early to late endosomes is achieved by relatively large (approximately 0.5 microm), long-lived and multivesicular intermediates, and their biogenesis depends on endosomal COP-I proteins. Here, we review recent work on the formation of these different transport intermediates, and we discuss, in particular, coat proteins, sorting signals contained in cargo molecules and the emerging role of lipid in vesicle biogenesis.     

DNA breaks as triggers for antigenic variation in African trypanosomes

The DNA repair machinery has been co-opted for antigenic variation in African trypanosomes. New work directly demonstrates that a double-strand break initiates a switch in the expressed variant surface coat.