African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

SummaryThe concept of specific chemotherapy was developed a century ago by Paul Ehrlich and others. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work 1,2, and the drugs that emerged remain in use for treating Human African Trypanosomiasis (HAT) 3. Ehrlich recognised the importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies, but these mechanisms have remained largely mysterious. Here, we use all five current HAT drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling identifies both known drug importers 4,5 and the only known pro-drug activator 6, and links more than fifty additional genes to drug action. A specific bloodstream stage invariant surface glycoprotein (ISG75) family mediates suramin uptake while the AP-1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and multiple putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of anti-trypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented levels of molecular insight into the mode of action of anti-trypanosomal drugs.

Lysosomal targeting of ubiquitylated endocytic cargo is mediated in part by the endosomal sorting complex required for transport (ESCRT) complexes, a system conserved between animals and fungi (Opisthokonta). Extensive comparative genomic analysis demonstrates that ESCRT factors are well conserved across the eukaryotic lineage and complexes I, II, III and III-associated are almost completely retained, indicating an early evolutionary origin. The conspicuous exception is ESCRT 0, which functions in recognition of ubiquitylated cargo, and is restricted to the Opisthokonta, suggesting that a distinct mechanism likely operates in the vast majority of eukaryotic organisms. Additional analysis suggests that ESCRT III and ESCRT III-associated components evolved through a concerted model. Functional conservation of the ESCRT system is confirmed by direct study in trypanosomes. Despite extreme sequence divergence, epitope-tagged ESCRT factors TbVps23 and TbVps28 localize to the endosomal pathway, placing the trypanosome multivesicular body (MVB) in juxtaposition to the early endosome and lysosome. Knockdown of TbVps23 partially prevents degradation of an ubiquitylated endocytosed transmembrane domain protein. Therefore, despite the absence of an ESCRT 0 complex, the trypanosome ESCRT/MVB system functions similarly to that of opisthokonts. Thus the ESCRT system is an ancient and well-conserved feature of eukaryotic cells but with key differences between diverse lineages.

The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.

Trypanosomes are important disease agents and excellent models for the study of evolutionary cell biology. The trypanosome flagellar pocket is a small invagination of the plasma membrane where the flagellum exits the cytoplasm and participates in many cellular processes. It is the only site of exocytosis and endocytosis and part of a multiorganelle complex that is involved in cell polarity and cell division. Several flagellar pocket-associated proteins have been identified and found to contribute to trafficking and virulence. In this Review we discuss the contribution of the flagellar pocket to protein trafficking, immune evasion and other processes.

SummaryThe presence of a nucleus and other membrane-bounded intracellular compartments is the defining feature of eukaryotic cells. Endosymbiosis accounts for the origins of mitochondria and plastids, but the evolutionary ancestry of the remaining cellular compartments is incompletely documented. Resolving the evolutionary history of organelle-identity encoding proteins within the endomembrane system is a necessity for unravelling the origins and diversification of the endogenously derived organelles. Comparative genomics reveals events after the last eukaryotic common ancestor (LECA), but resolution of events prior to LECA, and a full account of the intracellular compartments present in LECA, has proved elusive. We have devised and exploited a new phylogenetic strategy to reconstruct the history of the Rab GTPases, a key family of endomembrane-specificity proteins. Strikingly, we infer a remarkably sophisticated organellar composition for LECA, which we predict possessed as many as 23 Rab GTPases. This repertoire is significantly greater than that present in many modern organisms and unexpectedly indicates a major role for secondary loss in the evolutionary diversification of the endomembrane system. We have identified two Rab paralogues of unknown function but wide distribution, and thus presumably ancient nature; RabTitan and RTW. Furthermore, we show that many Rab paralogues emerged relatively suddenly during early metazoan evolution, which is in stark contrast to the lack of significant Rab family expansions at the onset of most other major eukaryotic groups. Finally, we reconstruct higher-order ancestral clades of Rabs primarily linked with endocytic and exocytic process, suggesting the presence of primordial Rabs associated with the establishment of those pathways and giving the deepest glimpse to date into pre-LECA history of the endomembrane system.

The emergence of an endomembrane system was a crucial stage in the prokaryote-to-eukaryote evolutionary transition. Recent genomic and molecular evolutionary analyses have provided insight into how this critical system arrived at its modern configuration. The apparent relative absence of prokaryotic antecedents for the endomembrane machinery contrasts with the situation for mitochondria, plastids and the nucleus. Overall, the evidence suggests an autogenous origin for the eukaryotic membrane-trafficking machinery. The emerging picture is that early eukaryotic ancestors had a complex endomembrane system, which implies that this cellular system evolved relatively rapidly after the proto-eukaryote diverged away from the other prokaryotic lines. Many of the components of the trafficking system are the result of gene duplications that have produced proteins that have similar functions but differ in their subcellular location. A proto-eukaryote possessing a very simple trafficking system could thus have evolved to near modern complexity in the last common eukaryotic ancestor (LCEA) via paralogous gene family expansion of the proteins encoding organelle identity. The descendents of this common ancestor have undergone further modification of the trafficking machinery; unicellular simplicity and multicellular complexity are the prevailing trend, but there are some remarkable counter-examples.