Control of Trypanosoma brucei brucei infections in tsetse, Glossina morsitans

Abstract Numbers of immature Trypanosoma brucei brucei within a tsetse midgut remain remarkably constant after establishment throughout the course of an infection, irrespective of whether the infection eventually matures. These results suggest a system of self regulation of the parasite population in the insect gut based on a form of programmed cell death which would carry advantages for both the parasite and the vector.     

Invariant surface proteins in bloodstream forms of Trypanosoma brucei

Antigenic variation of the glycoprotein forming the coat of African trypanosomes has been a dominant field of investigation for many years. The extravagant potential of these parasites to change their surface coat has destroyed hopes for a vaccine based on the variant surface glycoprotein. Recently, there has been a rising interest in the characterization of surface proteins that are not subject to antigenic variation. In this review, Peter Overath, Maliha Chaudhri, Dietmar Steverding and Karl Ziegelbauer summarize the present evidence for the occurrence, cellular localization and function of invariant surface proteins in Trypanosoma brucei.     

Endocytosis, membrane recycling and sorting of GPI-anchored proteins: Trypanosoma brucei as a model system

In the flagellated protozoon Trypanosoma brucei, endo- and exocytosis are restricted to a small area of the plasma membrane, the flagellar pocket. All endosomal compartments and the single Golgi complex are located within the posterior part of the cell between the flagellar pocket and the nucleus. The use of reverse genetic tools, including RNA interference, in combination with quantitative 3D-fluorescence and electron microscopic techniques has provided an insight into endosomal membrane traffic, which occurs at a very high rate and appears to exhibit a lower level of complexity than in mammalian cells. The flagellate is an excellent model system for studies on endocytosis, sorting and recycling of glycosylphosphatidylinositol-anchored glycoproteins, because 10(7) molecules of the variant surface glycoprotein form a dense coat at the cell's surface. Because the endocytic rate varies widely at different stages in the parasite's life cycle, trypanosomes may be used for investigating developmental aspects of their endocytic system.     

GPI-anchored proteins and free GPI glycolipids of procyclic form Trypanosoma brucei are nonessential for growth, are required for colonization of the tsetse fly, and are not the only components of the surface coat

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine-containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine-containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI-anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.     

Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.     

Trypanosoma brucei: TbRAB4 regulates membrane recycling and expression of surface proteins in procyclic forms

TbRAB4 is the Trypanosoma brucei orthologue of the small GTPase Rab4, which is implicated in the control of early endocytosis and recycling processes. TbRAB4 is expressed constitutively in the procyclic and bloodstream stages suggesting an important function throughout the trypanosome life-cycle. Previous work from our laboratory has shown TbRAB4 to be essential in the bloodstream form. Induction of double-stranded TbRAB4 RNA expression leads to a specific reduction in TbRAB4 protein levels and inhibition of growth in procyclic form T. brucei, with alterations in uptake and recycling as measured with the fluorophore FM4-64. Trypanosomes overexpressing GTP-locked TbRAB4(QL) mutants exhibit significant perturbations of endocytic and recycling pathways as well as disruption of surface expression of GPI-anchored proteins. Most significantly, both the endogenous GPI-anchored procyclins and an ectopically expressed GPI-anchored protein, the variant surface glycoprotein, are relocated from the surface to internal sites in TbRAB4 mutant cells. These data indicate that TbRAB4 is important in maintenance of normal surface expression of lipid-anchored proteins, and implicate recycling pathways as factors for modulation of surface protein expression in the procyclic trypanosome. The conservation of function of Rab4 throughout eukaryotic evolution demonstrated here indicates that the Rab4-mediated trafficking pathway is an extremely ancient component of the endocytic system.     

Trypanosoma brucei: two-dimensional gel analysis of the major glycosomal proteins during the life cycle

Kinetoplastid organisms possess a unique organelle, the glycosome, which compartmentalizes the Embden-Meyerhof segment of glycolysis and several other metabolic pathways. In Trypanosoma brucei many of the enzyme activities localized to the glycosome are stage regulated. Two-dimensional gel analysis was used to examine the characteristics, expression, and biosynthesis of the major glycosomal proteins. Two-dimensional gel maps of glycosomes from slender bloodforms and late intermediate-stumpy bloodforms (the precursors of procyclic forms) were indistinguishable, while those of procyclic form glycosomes showed extensive differences. Glycosomal phosphoenolpyruvate carboxykinase and malate dehydrogenase were identified to have subunit molecular weights of 60 and 34 kDa, respectively. We detected two hitherto undescribed glycosomal proteins, one of which is found only in bloodforms. All of the major proteins, except glucose phosphate isomerase, were highly basic. Stage regulation of glycosomal enzyme activities correlated with stage regulation of specific protein biosynthesis.     

The expression level determines the surface distribution of the transferrin receptor in Trypanosoma brucei

The transferrin receptor (TfR) of Trypanosoma brucei is a heterodimer attached to the surface membrane by a glycosylphosphatidylinositol (GPI) anchor. The TfR is restricted to the flagellar pocket, a deep invagination of the plasma membrane. The membrane of the flagellar pocket and the rest of the cell surface are continuous, and the mechanism that selectively retains the TfR in the pocket is unknown. Here, we report that the TfR is retained in the flagellar pocket by a specific and saturable mechanism. In bloodstream-form trypanosomes transfected with the TfR genes, TfR molecules escaped flagellar pocket retention and accumulated on the entire surface, even at modest (threefold) overproduction levels. Similar surface accumulation was observed when the TfR levels were physiologically upregulated threefold when trypanosomes were starved for transferrin. These results suggest that the TfR flagellar pocket retention mechanism is easily saturated and that control of the expression level is critical to maintain the restricted surface distribution of the receptor.     

Cyclical transmission of Trypanosoma brucei rhodesiense and Trypanosoma congolense by tsetse flies infected with culture-form procyclic trypanosomes

Culture procyclic forms of Trypanosoma brucei rhodesiense and Trypanosoma congolense were fed to Glossina morsitans morsitans through artificial membranes. A very high percentage of the flies so fed produced established midgut infections, a proportion of which went on to develop into mature metacyclic trypanosomes capable of infecting mammalian hosts. The method offers a safe, clean way of infecting tsetse flies with African trypanosomes which reduces the need for trypanosome-infected animals in the laboratory.     

Trypanosoma brucei brucei: variability in the association of some variant surface glycoproteins

In our isolation procedure, the surface antigens of the variants AnTat 1.1 and 1.10 (Trypanosoma brucei brucei) are essentially obtained as a disulfide-linked dimer while the AnTat 1.8 surface antigen is found as a mixture of monomer and disulfide-linked dimer. This observation may be related to the localization of the cysteine residues in the protein sequences. In the purification procedure using concanavalin-A Sepharose chromatography, besides the VSG elution by methyl-alpha-D-mannopyranoside, a quantitative elution of still bound VSG may be obtained by the addition of beta-mercaptoethanol to methyl-alpha-D-mannopyrannoside in the elution buffer. The surface antigen of the variant AnTat 1.1 was examined for molecular form at several different times during the release procedure. The disulfide-linked dimer could be observed within 30 min of the surface coat release, indicating its presence within the parasite.     

Arginine methylation regulates mitochondrial gene expression in Trypanosoma brucei through multiple effector proteins

Arginine methylation is a post-translational modification that impacts gene expression in both the cytoplasm and nucleus. Here, we demonstrate that arginine methylation also affects mitochondrial gene expression in the protozoan parasite, Trypanosoma brucei. Down-regulation of the major trypanosome type I protein arginine methyltransferase, TbPRMT1, leads to destabilization of specific mitochondrial mRNAs. We provide evidence that some of these effects are mediated by the mitochondrial RNA-binding protein, RBP16, which we previously demonstrated affects both RNA editing and stability. TbPRMT1 catalyzes methylation of RBP16 in vitro. Further, MALDI-TOF-MS analysis of RBP16 isolated from TbPRMT1-depleted cells indicates that, in vivo, TbPRMT1 modifies two of the three known methylated arginine residues in RBP16. Expression of mutated, nonmethylatable RBP16 in T. brucei has a dominant negative effect, leading to destabilization of a subset of those mRNAs affected by TbPRMT1 depletion. Our results suggest that the specificity and multifunctional nature of RBP16 are due, at least in part, to the presence of differentially methylated forms of the protein. However, some effects of TbPRMT1 depletion on mitochondrial gene expression cannot be accounted for by RBP16 action. Thus, these data implicate additional, unknown methylproteins in mitochondrial gene regulation.     

Trypanosoma brucei: release of variant surface glycoprotein during the parasite life cycle

African trypanosome variant surface antigen, which was released from the Trypanosoma brucei parasite at two stages in its life cycle, has been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. Variant surface antigen released as exoantigen into the bloodstream of infected rats resembled the soluble form of the surface antigen. Variant surface antigen released from parasites undergoing transformation to the uncoated procyclic stage was detected as two molecular species: soluble variant surface antigen and a cleavage product of variant surface antigen. The data presented are consistent with enzymatic cleavage of the variant surface antigen C-terminal hydrophobic moiety operating to release parasite surface coat from living parasites.