Trypanosoma brucei is one of only a few unicellular pathogens that thrives extracellularly in the vertebrate host. Consequently, the cell surface plays a critical role in both immune recognition and immune evasion. The variant surface glycoprotein (VSG) coats the entire surface of the parasite and acts as a flexible shield to protect invariant proteins against immune recognition. Antigenic variation of the VSG coat is the major virulence mechanism of trypanosomes. In addition, incessant motility of the parasite contributes to its immune evasion, as the resulting fluid flow on the cell surface drags immunocomplexes toward the flagellar pocket, where they are internalized. The flagellar pocket is the sole site of endo- and exocytosis in this organism. After internalization, VSG is rapidly recycled back to the surface, whereas host antibodies are thought to be transported to the lysosome for degradation. For this essential step to work, effective machineries for both sorting and recycling of VSGs must have evolved in trypanosomes. Our understanding of the mechanisms behind VSG recycling and VSG secretion, is by far not complete. This review provides an overview of the trypanosome secretory and endosomal pathways. Longstanding questions are pinpointed that, with the advent of novel technologies, might be answered in the near future.

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The use of glycosylphosphatidylinositol (GPI) to anchor proteins to the cell surface is widespread among eukaryotes. The GPI-anchor is covalently attached to the C-terminus of a protein and mediates the protein’s attachment to the outer leaflet of the lipid bilayer. GPI-anchored proteins have a wide range of functions, including acting as receptors, transporters, and adhesion molecules. In unicellular eukaryotic parasites, abundantly expressed GPI-anchored proteins are major virulence factors, which support infection and survival within distinct host environments. While, for example, the variant surface glycoprotein (VSG) is the major component of the cell surface of the bloodstream form of African trypanosomes, procyclin is the most abundant protein of the procyclic form which is found in the invertebrate host, the tsetse fly vector. Trypanosoma cruzi, on the other hand, expresses a variety of GPI-anchored molecules on their cell surface, such as mucins, that interact with their hosts. The latter is also true for Leishmania, which use GPI anchors to display, amongst others, lipophosphoglycans on their surface. Clearly, GPI-anchoring is a common feature in trypanosomatids and the fact that it has been maintained throughout eukaryote evolution indicates its adaptive value. Here, we explore and discuss GPI anchors as universal evolutionary building blocks that support the great variety of surface molecules of trypanosomatids.

All endo- and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the cell's single flagellum. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, may affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of the hook complex component TbMORN1 resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form Trypanosoma brucei and was found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement, and impaired access to the pocket membrane by surface-bound cargo. Inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket.

Background: Parasites of the order Trypanosomatida are known due to their medical relevance. Trypanosomes cause African sleeping sickness and Chagas disease in South America, and Leishmania Ross, 1903 species mutilate and kill hundreds of thousands of people each year. However, human pathogens are very few when compared to the great diversity of trypanosomatids. Despite the progresses made in the past decades on understanding the evolution of this group of organisms, there are still many open questions which require robust phylogenetic markers to increase the resolution of trees. Methods: Using two known 18S rDNA template structures (from Trypanosoma cruzi Chagas, 1909 and Trypanosoma brucei Plimmer & Bradford, 1899), individual 18S rDNA secondary structures were predicted by homology modeling. Sequences and their secondary structures, automatically encoded by a 12-letter alphabet (each nucleotide with its three structural states, paired left, paired right, unpaired), were simultaneously aligned. Sequence-structure trees were generated by neighbor joining and/or maximum likelihood.Results: With a few exceptions, all nodes within a sequence-structure maximum likelihood tree of 43 representative 18S rDNA sequence-structure pairs are robustly supported (bootstrap support >75). Even a quick and easy sequence-structure neighbor-joining analysis yields accurate results and enables reconstruction and discussion of the big picture for all 240 18S rDNA sequence-structure pairs of trypanosomatids that are currently available.Conclusions: We reconstructed the phylogeny of a comprehensive sampling of trypanosomes evaluated in the context of trypanosomatid diversity, demonstrating that the simultaneous use of 18S rDNA sequence and secondary structure data can reconstruct robust phylogenetic trees.

Since the observation of the great pleomorphism of fish trypanosomes, in vitro culture has become an important tool to support taxonomic studies investigating the biology of cultured parasites, such as their structure, growth dynamics, and cellular cycle. Relative to their biology, ex vivo and in vitro studies have shown that these parasites, during the multiplication process, duplicate and segregate the kinetoplast before nucleus replication and division. However, the inverse sequence (the nucleus divides before the kinetoplast) has only been documented for a species of marine fish trypanosomes on a single occasion. Now, this previously rare event was observed in Trypanosoma abeli, a freshwater fish trypanosome. Specifically, from 376 cultured parasites in the multiplication process, we determined the sequence of organelle division for 111 forms; 39% exhibited nucleus duplication prior to kinetoplast replication. Thus, our results suggest that nucleus division before the kinetoplast may not represent an accidental or erroneous event occurring in the main pathway of parasite reproduction, but instead could be a species-specific process of cell biology in trypanosomes, such as previously noticed for Leishmania. This "alternative" pathway for organelle replication is a new field to be explored concerning the biology of marine and freshwater fish trypanosomes.

The variable regions (V1–V9) of the 18S rDNA are routinely used in barcoding and phylogenetics. In handling these data for trypanosomes, we have noticed a misunderstanding that has apparently taken a life of its own in the literature over the years. In particular, in recent years, when studying the phylogenetic relationship of trypanosomes, the use of V7/V8 was systematically established. However, considering the current numbering system for all other organisms (including other Euglenozoa), V7/V8 was never used. In Maia da Silva et al. [Parasitology 2004, 129, 549–561], V7/V8 was promoted for the first time for trypanosome phylogenetics, and since then, more than 70 publications have replicated this nomenclature and even discussed the benefits of the use of this region in comparison to V4. However, the primers used to amplify the variable region of trypanosomes have actually amplified V4 (concerning the current 18S rDNA numbering system).

Background: Parasites of the order Trypanosomatida are known due to their medical relevance. Trypanosomes cause African sleeping sickness and Chagas disease in South America, and Leishmania ROSS, 1903 species mutilate and kill hundreds of thousands of people each year. However, human pathogens are very few when compared to the great diversity of trypanosomatids. Despite the progresses made in the past decades on understanding the evolution of this group of organisms, there are still many open questions which require robust phylogenetic markers to increase the resolution of trees. Methods: Using two known 18S rDNA template structures (from Trypanosoma cruzi CHAGAS, 1909 and Trypanosoma brucei PLIMMER & BRADFORD, 1899), individual 18S rDNA secondary structures were predicted by homology modeling. Sequences and their secondary structures, automatically encoded by a 12-letter alphabet (each nucleotide with its three structural states, paired left, paired right, unpaired), were simultaneously aligned. Sequence-structure trees were generated by neighbor joining and/or maximum likelihood. Results: With a few exceptions, all nodes within a sequence-structure maximum likelihood tree of 43 representative 18S rDNA sequence-structure pairs are robustly supported (bootstrap support >75). Even a quick and easy sequence-structure neighbor-joining analysis yields accurate results and enables reconstruction and discussion of the big picture for all 240 18S rDNA sequence-structure pairs of trypanosomatids that are currently available. Conclusions: We reconstructed the phylogeny of a comprehensive sampling of trypanosomes evaluated in the context of trypanosomatid diversity, demonstrating that the simultaneous use of 18S rDNA sequence and secondary structure data can reconstruct robust phylogenetic trees.