The evolutionary history of kinetoplastids and their kinetoplasts

Despite extensive phylogenetic analysis of small subunit ribosomal RNA (SSUrRNA) genes, the deep-level relationships among kinetoplastids remain poorly understood, limiting our grasp of their evolutionary history, especially the origins of their bizarre mitochondrial genome organizations. In this study we examine the SSUrRNA data in the light of a new marker--cytoplasmic heat shock protein 90 (hsp90) sequences. Our phylogenetic analyses divide kinetoplastids into four main clades. Clades 1-3 include the various bodonid kinetoplastids. Trypanosomatids comprise the fourth clade. SSUrRNA analyses give vastly different and poorly supported positions for the root of the kinetoplastid tree, depending on the out-group and analysis method. This is probably due to the extraordinary length of the branch between kinetoplastids and any out-group. In contrast, almost all hsp90 analyses place the root between clade 1 (including Dimastigella, Rhynchomonas, several Bodo spp., and probably Rhynchobodo) and all other kinetoplastids. Maximum likelihood and maximum likelihood distance analyses of hsp90 protein and second codon-position nucleotides place trypanosomatids adjacent to Bodo saltans and Bodo cf. uncinatus (clade 3), as (weakly) do SSUrRNA analyses. Hsp90 first codon- plus second codon-position nucleotide analyses return a slightly different topology. We show that this may be an artifact caused, in part, by the different evolutionary behavior of first- and second-codon positions. This study provides the most robust evidence to date that trypanosomatids are descended from within bodonids and that B. saltans is a close relative of trypanosomatids. A total reevaluation of the high-level systematics within kinetoplastids is needed. We confirm that the interlocking network organization of kinetoplast DNA seen in trypanosomatids is a derived condition within kinetoplastids but suggest that open-conformation minicircles may have arisen early in kinetoplastid evolution. Further understanding of the evolution of kinetoplast structure and RNA editing is hampered by a paucity of data from basal (i.e., clade 1) bodonids.     

Kinetoplastid phylogenetics, with special reference to the evolution of parasitic trypanosomes

To fully understand the evolutionary history of parasitic kinetoplastids and to understand the context within which the evolution of each parasite group has developed, an understanding not just of the parasites, but of all kinetoplastids is required. Accordingly, this paper provides an overview of kinetoplastid evolution and systematics, including coverage of the proposal by Moreira et al. (2004) to divide kinetoplasts into Prokinetoplastina (Ichthyobodo and Perkinsiella) and Metakinetoplastina (other bodonids and trypanosomatids). The implications of such a revision, with regard to correctly identifying outgroup taxa for studies of evolution within taxa of medical importance, are addressed, together with a more detailed review of the evolution and origins of the trypanosomes in the light of new phylogenies, new approaches and revisions in kinetoplastid systematics.     

Ribosomal RNA phylogeny of bodonid and diplonemid flagellates and the evolution of euglenozoa

Euglenozoa is a major phylum of excavate protozoa (comprising euglenoids, kinetoplastids, and diplonemids) with highly unusual nuclear, mitochondrial, and chloroplast genomes. To improve understanding of euglenozoan evolution, we sequenced nuclear small-subunit rRNA genes from 34 bodonids (Bodo, Neobodo, Parabodo, Dimastigella-like, Rhynchobodo, Rhynchomonas, and unidentified strains), nine diplonemids (Diplonema, Rhynchopus), and a euglenoid (Entosiphon). Phylogenetic analysis reveals that diplonemids and bodonids are more diverse than previously recognised, but does not clearly establish the branching order of kinetoplastids, euglenoids, and diplonemids. Rhynchopus is holophyletic; parasitic species arose from within free-living species. Kinetoplastea (bodonids and trypanosomatids) are robustly holophyletic and comprise a major clade including all trypanosomatids and most bodonids ('core bodonids') and a very divergent minor one including Ichthyobodo. The root of the major kinetoplastid clade is probably between trypanosomatids and core bodonids. Core bodonids have three distinct subclades. Clade 1 has two distinct Rhynchobodo-like lineages; a lineage comprising Dimastigella and Rhynchomonas; and another including Cruzella and Neobodo. Clade 2 comprises Cryptobia/ Trypanoplasma, Procryptobia, and Parabodo. Clade 3 is an extensive Bodo saltans species complex. Neobodo designis is a vast genetically divergent species complex with mutually exclusive marine and freshwater subclades. Our analysis supports three phagotrophic euglenoid orders: Petalomonadida (holophyletic), Ploeotiida (probably holophyletic), Peranemida (paraphyletic).     

Phylogeny of the kinetoplastida: taxonomic problems and insights into the evolution of parasitism

To further investigate phylogeny of kinetoplastid protozoa, the sequences of small subunit (18S) ribosomal RNA of nine bodonid isolates and ten isolates of insect trypanosomatids have been determined. The root of the kinetoplastid tree was attached to the branch of Bodo designis and/or Cruzella marina. The suborder Trypanosomatina appeared as a monophyletic group, while the suborder Bodonina was paraphyletic. Among bodonid lineages, parasitic organisms were intermingled with free-living ones, implying multiple transitions to parasitism and supporting the 'vertebrate-first hypothesis'. The tree indicated that the genera Cryptobia and Bodo are artificial taxa. Separation of fish cryptobias and Trypanoplasma borreli as different genera was not supported. In trypanosomatids, the genera Leptomonas and Blastocrithidia were polyphyletic, similar to the genera Herpetomonas and Crithidia and in contrast to the monophyletic genera Trypanosoma and Phytomonas. This analysis has shown that the morphological classification of kinetoplastids does not in general reflect their genetic affinities and needs a revision.     

Evolutionary analysis of synteny and gene fusion for pyrimidine biosynthetic enzymes in Euglenozoa: an extraordinary gap between kinetoplastids and diplonemids

A unique feature of the genome architecture in the parasitic trypanosomatid protists is large-scale synteny. We addressed the evolutionary trait of synteny in the eukaryotic group, Euglenozoa, which consists of euglenoids (earliest branching), diplonemids, and kinetoplastids (trypanosomatids and bodonids). Synteny of the pyrimidine biosynthetic (pyr) gene cluster, which constitutes part of a large syntenic cluster in trypanosomatids and includes four separate genes (pyr1-pyr4) and one fused gene (pyr6/pyr5 fusion), was conserved in the bodonid, Parabodo caudatus. In the diplonemid, Diplonema papillatum, we identified pyr4 and pyr6 genes. Phylogenetic analyses of pyr4 and pyr6 showed the separate origin of each in kinetoplastids and euglenoids/diplonemids and suggested that kinetoplastids have acquired these genes via lateral gene transfer (LGT). Because replacement of genes by non-orthologs within the syntenic cluster is highly unlikely, we concluded that, after separation of the line leading to diplonemids, the syntenic pyr gene cluster was established in the common ancestor of kinetoplastids, preceded by their acquisition via LGT. Notably, we found that diplonemid pyr6 is a stand-alone gene, inconsistent with both euglenoid pyr5/pyr6 and kinetoplastid pyr6/pyr5 fusions. Our findings provide insights into the evolutionary gaps within Euglenozoa and the evolutionary trait of rearrangement of gene fusion in this lineage.     

Early evolution within kinetoplastids (euglenozoa), and the late emergence of trypanosomatids

Many important relationships amongst kinetoplastids, including the position of trypanosomatids, remain uncertain, with limited taxon sampling of markers other than small subunit ribosomal RNA (SSUrRNA). We report gene sequences for cytosolic heat shock proteins 90 and/or 70 (HSP90, HSP70) from the potentially early-diverging kinetoplastids Ichthyobodo necator and Rhynchobodo sp., and from bodonid clades ‘2’ (Parabodonidae) and ‘3’ (Eubodonidae). Some of the new cytosolic HSP70 sequences represent a distinct paralog family (HSP70-B), which is related to yet another paralog known from trypanosomatids (HSP70-C). The (HSP70-B, HSP70-C) clade seemingly diverged before the separation between kinetoplastids and diplonemids. Protein phylogenies support the basal placement of Ichthyobodo within kinetoplastids. Unexpectedly, Rhynchobodo usually forms the next most basal group, separated from the clade ‘1’ bodonids with which it has been allied. Bootstrap support is often weak, but the possibility that Rhynchobodo represents a separate early-diverging lineage within core kinetoplastids deserves further testing. Trypanosomatids always fall remote from the root of kinetoplastids, forming a specific relationship with bodonid clades 2 (and 3), generally with strong bootstrap support. These protein trees with improved taxon sampling provide the best evidence to date for a ‘late’ emergence of trypanosomatids, contradicting recent SSUrRNA-based proposals for a relatively early divergence of this group.     

Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids

Kinetoplastids are a large group of free-living and parasitic eukaryotic flagellates, including the medically important trypanosomatids (e.g., Trypanosoma and Leishmania) and the widespread free-living and parasitic bodonids. Small subunit rRNA- and conserved protein-based phylogenies support the division of kinetoplastids into five orders (Prokinetoplastida, Neobodonida, Parabodonida, Eubodonida, and Trypanosomatida), but they produce incongruent results regarding their relative branching order, in particular for the position of the Trypanosomatida. In general, small subunit rRNA tends to support their early emergence, whereas protein phylogenies most often support a more recent origin from within bodonids. In order to resolve this question through a phylogenomic approach, we carried out massive parallel sequencing of cDNA from representatives of three bodonid orders (Bodo saltans -Eubodonida-, Procryptobia sorokini -Parabodonida-, and Rhynchomonas nasuta -Neobodonida-). We identified 64 well-conserved proteins shared by these species, four trypanosomatids, and two closely related outgroup species (Euglena gracilis and Diplonema papillatum). Phylogenetic analysis of a concatenated data set yielded a strongly supported tree showing the late emergence of trypanosomatids as a sister group of the Eubodonida. In addition, we identified homologues of proteins involved in trypanosomatid mitochondrial mRNA editing in the three bodonid species, suggesting that editing may be widespread in kinetoplastids. Comparison of expressed sequences from mitochondrial genes showed variability at U positions, in agreement with the existence of editing activity in the three bodonid orders most closely related to trypanosomatids (Neobodonida, Parabodonida, and Eubodonida). Mitochondrial mRNA editing appears to be an ancient phenomenon in kinetoplastids.     

Occurrence of multiple, independent gene fusion events for the fifth and sixth enzymes of pyrimidine biosynthesis in different eukaryotic groups

The genes encoding orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzymes in the de novo pyrimidine biosynthetic pathway, are fused as OPRT-OMPDC in most eukaryotic groups. On the other hand, the inversely linked OMPDC-OPRT fusion is present in trypanosomatids, belonging to kinetoplastids together with bodonids in a supergroup, Euglenozoa. Here, we show the presence of OMPDC-OPRT in the bodonid, Bodo caudatus, while OPRT-OMPDC in Euglena gracilis, another euglenozoan species belonging to euglenoids. These results suggest that the OMPDC-OPRT fusion event occurred in a common ancestor of kinetoplastids. Genome sequence database searches further revealed the presence of OMPDC-OPRT in stramenopiles and cyanobacteria. Phylogenetic reconstruction of OPRT and OMPDC rejected statistically the monophyly of the OPRT domains of stramenopile and kinetoplastid OMPDC-OPRT, demonstrating that these gene fusions do not share a common evolutionary origin, despite the identical gene order. Thus, the OMPDC-OPRT fusion is likely to have emerged independently in these eukaryotic groups. Phylogenetic analyses also suggested that cyanobacterial OMPDC-OPRT arose via lateral transfer. We conclude that gene fusion events occur more frequently than previously thought and that lateral gene transfer has made a marked contribution to establishment of the rearranged structure of OPRT and OMPDC genes in eukaryotes.     

The Origin of Dihydroorotate Dehydrogenase Genes of Kinetoplastids, with Special Reference to Their Biological Significance and Adaptation to Anaerobic, Parasitic Conditions

Trypanosoma cruzi dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is localized in the cytosol and utilizes fumarate as electron acceptor (fumarate reductase activity), while the enzyme from other various eukaryotes is mitochondrial membrane-linked. Here we report that DHOD-knockout T. cruzi did not express the enzyme protein and could not survive even in the presence of pyrimidine nucleosides, substrates for the potentially active salvage pathway, suggesting a vital role of fumarate reductase activity in the regulation of cellular redox balance. Cloning and phylogenetic analysis of euglenozoan DHOD genes showed that the euglenoid Euglena gracilis had a mitochondrial DHOD and that biflagellated bodonids, a sister group of trypanosomatids within kinetoplastids, harbor the cytosolic DHOD. Further, Bodo saliens, a bodonid, had an ACT/DHOD gene fusion encoding aspartate carbamoyltransferase (ACT), the second enzyme of the de novo pyrimidine pathway, and DHOD. This is the first report of this novel gene structure. These results are consistent with suggestions that an ancient common ancestor of Euglenozoa had a mitochondrial DHOD whose descendant exists in E. gracilis and that a common ancestor of kinetoplastids (bodonids and trypanosomatids) subsequently acquired a cytosolic DHOD by horizontal gene transfer. The cytosolic DHOD gene thus acquired may have contributed to adaptation to anaerobiosis in the kinetoplastid lineage and further contributed to the subsequent establishment of parasitism in a trypanosomatid ancestor. Different molecular strategies for anaerobic adaptation in pyrimidine biosynthesis, used by kinetoplastids and by euglenoids, are discussed. Evolutionary implications of the ACT/DHOD gene fusion are also discussed.Sequence availability: The nucleotide sequence data reported here appear in the GenBank, EMBL, and DDBJ databases with the accession numbers AB120414, AB159227, and AB159228 for Euglena gracilis dihydroorotate dehydrogenase (DHOD), Bodo saliens aspartate carbamoyltransferase/dihydroorotate dehydrogenase (ACT/DHOD), and B. caudatus DHOD, respectively.Reviewing Editor: Dr. Patrick Keeling     

Compartmentalization of a glycolytic enzyme in Diplonema, a non-kinetoplastid euglenozoan

Glycosomes are peroxisome-related organelles containing glycolytic enzymes that have been found only in kinetoplastids. We show here that a glycolytic enzyme is compartmentalized in diplonemids, the sister group of kinetoplastids. We found that, similar to kinetoplastid aldolases, the fructose 1,6-bisphosphate aldolase of Diplonema papillatum possesses a type 2-peroxisomal targeting signal. Western blotting showed that this aldolase was present predominantly in the membrane/organellar fraction. Immunofluorescence analysis showed that this aldolase had a scattered distribution in the cytosol, suggesting its compartmentalization. In contrast, orotidine-5'-monophosphate decarboxylase, a non-glycolytic glycosomal enzyme in kinetoplastids, was shown to be a cytosolic enzyme in D. papillatum. Since euglenoids, the earliest diverging branch of Euglenozoa, do not possess glycolytic compartments, these findings suggest that the routing of glycolytic enzymes into peroxisomes may have occurred in a common ancestor of diplonemids and kinetoplastids, followed by diversification of these newly established organelles in each of these euglenozoan lineages.     

cAMP signalling in the kinetoplastid protozoa

Several species of kinetoplastid protozoa cause major human infectious diseases. Trypanosoma cruzi is responsible for the fatal Chagas disease in large parts of South America, the various species of Leishmania cause a number of different human diseases with millions of patients world-wide, and the African trypanosome Trypanosoma brucei is the agent of human sleeping sickness, a disastrously re-emerging epidemic of fatal infections in Sub-Saharan Africa. Chemotherapy of all of these infections is in a very unsatisfactory state. cAMP signalling pathways in humans have provided interesting drug targets for a number of clinical conditions, from asthma to impotency. Similarly, cAMP signalling in kinetoplastids might offer useful targets for the development of novel antiparasitic drugs, which makes their exploration an urgent need. Current knowledge suggests that cAMP signalling proceeds along very similar pathways in all kinetoplastid pathogens (T. cruzi, the Leishmanias and T. brucei). Their adenylyl cyclases are structurally very different from the human enzymes and appear to function as enzyme-linked cell surface receptors. They might represent the major sensory apparatus of the kinetoplastids, guiding much of their environmental sensing and host/parasite interaction. The cAMP-specific phosphodiesterases of the kinetoplastids are rather similar to those of human cells and might function in similar ways. Essentially nothing is known on downstream effectors of cAMP in the kinetoplastids. Homologues of protein kinase A and its regulatory subunits have been identified, but their biochemical properties seem to be disctinct from that of mammalian protein kinase A.     

Group-Specific PCR Primers to Amplify 24S a-Subunit rRNA Genes from Kinetoplastida (Protozoa) Used in Denaturing Gradient Gel Electrophoresis

We developed and tested a set of primers for amplification of a region of the 24S a-subunit rRNA genes (24S rDNA) specific to Kinetoplastida (Protozoa). The reverse primer was supplied with a GC rich region in the 5? end in order to make the PCR product suitable for analysis by denaturing gradient gel electrophoresis (DGGE). PCR product was obtained from all the kinetoplastids tested and no PCR product was obtained from any other Eukaryotes or Prokaryotes tested. It was possible to distinguish between all pure cultures of kinetoplastids by denaturing gradient gel electrophoresis in gels ranging from 20% to 60% denaturants. PCR-DGGE analysis of DNA purified from lake sediment revealed approximately 20 bands indicating high kinetoplastid diversity. Direct cloning and sequencing of 24S rDNA sequences retrieved from the lake sediment by PCR also showed high kinetoplastid diversity. Of 43 clones, 27 different sequences were found. Alignments and phylogenetic analysis showed that a majority of the sequences were most closely related to the Bodonidae. Four sequences were closer to the Trypanosomatidae, whereas three sequences fell outside both groups. The PCR-DGGE procedure developed in this study has been shown to be useful for distinguishing between different kinetoplastid species. Thus, it may be a useful tool for evaluating the genetic diversity of this group in environmental samples, e.g., as a result of perturbation. Another possible application of this method is in fast and accurate screening for the presence and identification of pathological parasitic Kinetoplastida from environmental samples and for diagnostics of human and animal infections.     

Cryptic paraflagellar rod in endosymbiont-containing kinetoplastid protozoa

Cilia and flagella are central to many biological processes in a diverse range of organisms. The kinetoplastid protozoa are very appealing models for the study of flagellar function, particularly in the light of the availability of extensive trypanosomatid genome information. In addition to the highly conserved 9 + 2 axoneme, the kinetoplastid flagellum contains a characteristic paraflagellar rod structure (PFR). The PFR is necessary for full motility and provides support for metabolic regulators that may influence flagellar beating. However, there is an intriguing puzzle: one clade of endosymbiont-containing kinetoplastids apparently lack a PFR yet are as motile as species that possess a PFR and are able to attach to the invertebrate host epithelia. We investigated how these organisms are able to locomote despite the apparent lack of PFR. Here we have identified a PFR1 gene in the endosymbiont-bearing trypanosome Crithidia deanei. This gene is expressed in C. deanei and is able to partially complement a pfr1 null mutation in Leishmania mexicana cells, demonstrating that the encoded protein is functional. Careful reexamination of C. deanei flagellar ultrastructure revealed a greatly reduced PFR missed by many previous analyses. This affirms the PFR as a canonical organelle of kinetoplastids. Moreover, although PFR proteins have been conserved in evolution, primary sequence differences contribute to particular PFR morphotypes characteristic of different kinetoplastid species.     

MOLECULAR EVOLUTION OF EUGLENOZOAN PARAXONEMAL ROD GENES par1 AND par2 COINCIDES WITH PHYLOGENETIC RECONSTRUCTION BASED ON SMALL SUBUNIT rDNA DATA1

Emergent flagella of Euglenozoa consist of two prominent structural elements: the axoneme built by microtubules with motor proteins to enable the movement of the flagellum and a highly organized protein structure of unknown function, called the paraxonemal rod (PAR), which consists of two major proteins paralleling the axoneme of euglenid and kinetoplastid emergent flagella. These flagellar structures are considered apomorphic characters of Euglenozoa. We examined the evolution of the genes par1 and par2 encoding the two major proteins, where we could show that these proteins are encoded by two very similar genes found in kinetoplastids and euglenids. The branching pattern indicated a gene duplication before the diversification into euglenids and kinetoplastids. In the clades of the genes, subtrees of euglenid and kinetoplastid monophyla arose. Both genes showed strong genetic diversity with biased GC content at taxon rather than at gene level. We also examined phylogenies inferred from PAR genes that are well in agreement with established small subunit rDNA analyses. Both showed further separation of the euglenid subtree into primary osmotrophs and a phototrophic clade, including secondarily derived osmotrophs.     

Protein trafficking in kinetoplastid protozoa.

The kinetoplastid protozoa infect hosts ranging from invertebrates to plants and mammals, causing diseases of medical and economic importance. They are the earliest-branching organisms in eucaryotic evolution to have either mitochondria or peroxisome-like microbodies. Investigation of their protein trafficking enables us to identify characteristics that have been conserved throughout eucaryotic evolution and also reveals how far variations, or alternative mechanisms, are possible. Protein trafficking in kinetoplastids is in many respects similar to that in higher eucaryotes, including mammals and yeasts. Differences in signal sequence specificities exist, however, for all subcellular locations so far examined in detail--microbodies, mitochondria, and endoplasmic reticulum--with signals being more degenerate, or shorter, than those of their higher eucaryotic counterparts. Some components of the normal array of trafficking mechanisms may be missing in most (if not all) kinetoplastids: examples are clathrin-coated vesicles, recycling receptors, and mannose 6-phosphate-mediated lysosomal targeting. Other aspects and structures are unique to the kinetoplastids or are as yet unexplained. Some of these peculiarities may eventually prove to be weak points that can be used as targets for chemotherapy; others may turn out to be much more widespread than currently suspected.