Lexis and Grammar of Mitochondrial RNA Processing in Trypanosomes.

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.

Separating the Wheat from the Chaff: RNA Editing and Selection of Translatable mRNA in Trypanosome Mitochondria.

In the mitochondria of trypanosomes and related kinetoplastid protists, most mRNAs undergo a long and sophisticated maturation pathway before they can be productively translated by mitochondrial ribosomes. Some of the aspects of this pathway (identity of the promotors, transcription initiation, and termination signals) remain obscure, and some (post-transcriptional modification by U-insertion/deletion, RNA editing, 3'-end maturation) have been illuminated by research during the last decades. The RNA editing creates an open reading frame for a productive translation, but the fully edited mRNA often represents a minor fraction in the pool of pre-edited and partially edited precursors. Therefore, it has been expected that the final stages of the mRNA processing generate molecular hallmarks, which allow for the efficient and selective recognition of translation-competent templates. The general contours and several important details of this process have become known only recently and represent the subject of this review.

Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host.

In this review, we summarize the current knowledge concerning the eukaryotic protozoan parasite Leishmania tarentolae, with a main focus on its potential for biotechnological applications. We will also discuss the genus, subgenus, and species-level classification of this parasite, its life cycle and geographical distribution, and similarities and differences to human-pathogenic species, as these aspects are relevant for the evaluation of biosafety aspects of L. tarentolae as host for recombinant DNA/protein applications. Studies indicate that strain LEM-125 but not strain TARII/UC of L. tarentolae might also be capable of infecting mammals, at least transiently. This could raise the question of whether the current biosafety level of this strain should be reevaluated. In addition, we will summarize the current state of biotechnological research involving L. tarentolae and explain why this eukaryotic parasite is an advantageous and promising human recombinant protein expression host. This summary includes overall biotechnological applications, insights into its protein expression machinery (especially on glycoprotein and antibody fragment expression), available expression vectors, cell culture conditions, and its potential as an immunotherapy agent for human leishmaniasis treatment. Furthermore, we will highlight useful online tools and, finally, discuss possible future applications such as the humanization of the glycosylation profile of L. tarentolae or the expression of mammalian recombinant proteins in amastigote-like cells of this species or in amastigotes of avirulent human-pathogenic Leishmania species.

Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution.

Unicellular flagellates of the family Trypanosomatidae are obligatory parasites of invertebrates, vertebrates and plants. Dixenous species are aetiological agents of a number of diseases in humans, domestic animals and plants. Their monoxenous relatives are restricted to insects. Because of the high biological diversity, adaptability to dramatically different environmental conditions, and omnipresence, these protists have major impact on all biotic communities that still needs to be fully elucidated. In addition, as these organisms represent a highly divergent evolutionary lineage, they are strikingly different from the common 'model system' eukaryotes, such as some mammals, plants or fungi. A number of excellent reviews, published over the past decade, were dedicated to specialized topics from the areas of trypanosomatid molecular and cell biology, biochemistry, host-parasite relationships or other aspects of these fascinating organisms. However, there is a need for a more comprehensive review that summarizing recent advances in the studies of trypanosomatids in the last 30 years, a task, which we tried to accomplish with the current paper.

Trypanosomatids Are Much More than Just Trypanosomes: Clues from the Expanded Family Tree.

Trypanosomes and leishmanias are widely known parasites of humans. However, they are just two out of several phylogenetic lineages that constitute the family Trypanosomatidae. Although dixeny - the ability to infect two hosts - is a derived trait of vertebrate-infecting parasites, the majority of trypanosomatids are monoxenous. Like their common ancestor, the monoxenous Trypanosomatidae are mostly parasites or commensals of insects. This review covers recent advances in the study of insect trypanosomatids, highlighting their diversity as well as genetic, morphological and biochemical complexity, which, until recently, was underappreciated. The investigation of insect trypanosomatids is providing an important foundation for understanding the origin and evolution of parasitism, including colonization of vertebrates and the appearance of human pathogens.

RSM22, mtYsxC and PNKD-like proteins are required for mitochondrial translation in Trypanosoma brucei.

Mitochondrial ribosomes evolved from prokaryotic ribosomes, with which they therefore share more common features than with their counterparts in the cytosol. Yet, mitochondrial ribosomes are highly diverse in structure and composition, having undergone considerable changes, including reduction of their RNA component and varying degree of acquisition of novel proteins in various phylogenetic lineages. Here, we present functional analysis of three putative mitochondrial ribosome-associated proteins (RSM22, mtYsxC and PNKD-like) in Trypanosoma brucei, originally identified by database mining. While in other systems the homologs of RSM22 are known as components of mitochondrial ribosomes, YsxC was linked with ribosomes only in bacteria. The PNKD-like protein shows similarity to a human protein, the defects of which cause PNKD (paroxysmal non-kinesigenic dyskinesia). Here we show that all three proteins are important for the growth of T. brucei. They play an important function in mitochondrial translation, as their ablation by RNAi rapidly and severely affected the de novo synthesis of mitochondrial proteins. Moreover, following the RNAi-mediated depletion of RSM22, structure of the small subunit of mitochondrial ribosome becomes severely compromised, suggesting a role of RSM22 in ribosomal assembly and/or stability.

Structures and stabilization of kinetoplastid-specific split rRNAs revealed by comparing leishmanial and human ribosomes.

The recent success in ribosome structure determination by cryoEM has opened the door to defining structural differences between ribosomes of pathogenic organisms and humans and to understand ribosome-targeting antibiotics. Here, by direct electron-counting cryoEM, we have determined the structures of the Leishmania donovani and human ribosomes at 2.9 Å and 3.6 Å, respectively. Our structure of the leishmanial ribosome elucidates the organization of the six fragments of its large subunit rRNA (as opposed to a single 28S rRNA in most eukaryotes, including humans) and reveals atomic details of a unique 20 amino acid extension of the uL13 protein that pins down the ends of three of the rRNA fragments. The structure also fashions many large rRNA expansion segments. Direct comparison of our human and leishmanial ribosome structures at the decoding A-site sheds light on how the bacterial ribosome-targeting drug paromomycin selectively inhibits the eukaryotic L. donovani, but not human, ribosome.

Genome of Leptomonas pyrrhocoris: a high-quality reference for monoxenous trypanosomatids and new insights into evolution of Leishmania.

Many high-quality genomes are available for dixenous (two hosts) trypanosomatid species of the genera Trypanosoma, Leishmania, and Phytomonas, but only fragmentary information is available for monoxenous (single-host) trypanosomatids. In trypanosomatids, monoxeny is ancestral to dixeny, thus it is anticipated that the genome sequences of the key monoxenous parasites will be instrumental for both understanding the origin of parasitism and the evolution of dixeny. Here, we present a high-quality genome for Leptomonas pyrrhocoris, which is closely related to the dixenous genus Leishmania. The L. pyrrhocoris genome (30.4 Mbp in 60 scaffolds) encodes 10,148 genes. Using the L. pyrrhocoris genome, we pinpointed genes gained in Leishmania. Among those genes, 20 genes with unknown function had expression patterns in the Leishmania mexicana life cycle suggesting their involvement in virulence. By combining differential expression data for L. mexicana, L. major and Leptomonas seymouri, we have identified several additional proteins potentially involved in virulence, including SpoU methylase and U3 small nucleolar ribonucleoprotein IMP3. The population genetics of L. pyrrhocoris was also addressed by sequencing thirteen strains of different geographic origin, allowing the identification of 1,318 genes under positive selection. This set of genes was significantly enriched in components of the cytoskeleton and the flagellum.

Novel Trypanosomatid-Bacterium Association: Evolution of Endosymbiosis in Action.

UNLABELLED: We describe a novel symbiotic association between a kinetoplastid protist, Novymonas esmeraldas gen. nov., sp. nov., and an intracytoplasmic bacterium, "Candidatus Pandoraea novymonadis" sp. nov., discovered as a result of a broad-scale survey of insect trypanosomatid biodiversity in Ecuador. We characterize this association by describing the morphology of both organisms, as well as their interactions, and by establishing their phylogenetic affinities. Importantly, neither partner is closely related to other known organisms previously implicated in eukaryote-bacterial symbiosis. This symbiotic association seems to be relatively recent, as the host does not exert a stringent control over the number of bacteria harbored in its cytoplasm. We argue that this unique relationship may represent a suitable model for studying the initial stages of establishment of endosymbiosis between a single-cellular eukaryote and a prokaryote. Based on phylogenetic analyses, Novymonas could be considered a proxy for the insect-only ancestor of the dixenous genus Leishmania and shed light on the origin of the two-host life cycle within the subfamily Leishmaniinae. IMPORTANCE: The parasitic trypanosomatid protist Novymonas esmeraldas gen. nov., sp. nov. entered into endosymbiosis with the bacterium "Ca. Pandoraea novymonadis" sp. nov. This novel and rather unstable interaction shows several signs of relatively recent establishment, qualifying it as a potentially unique transient stage in the increasingly complex range of eukaryotic-prokaryotic relationships.

Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes.

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3' adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation.

Host-specificity of Monoxenous Trypanosomatids: Statistical Analysis of the Distribution and Transmission Patterns of the Parasites from Neotropical Heteroptera.

Host-parasite relationships and parasite biodiversity have been the center of attention for many years; however the primary data obtained from large-scale studies remain scarce. Our long term investigations of trypanosomatid (Euglenozoa: Kinetoplastea) biodiversity from Neotropical Heteroptera have yielded almost one hundred typing units (TU) of trypanosomatids from one hundred twenty host species. Half of the parasites' TUs were documented in a single host species only but the rest were found parasitizing two to nine species of hosts, with logarithmic distribution best describing the observed distribution of parasites among hosts. Different host superfamilies did not show significant differences in numbers of trypanosomatid TUs they carry, with exception of Pyrrhocoroidea which showed higher parasite richness than any other group tested. Predatory reduviids shared significantly larger numbers of parasite TUs with phytophagous mirids and coreids than the numbers shared between any other groups. These results show that the specificity of trypanosomatid-heteropteran associations is not very strict: parasites seem to be transmissible between different host groups within the same niche and predatory hosts may acquire parasites from their prey.

U-insertion/deletion RNA editing multiprotein complexes and mitochondrial ribosomes in Leishmania tarentolae are located in antipodal nodes adjacent to the kinetoplast DNA.

We studied the intramitochondrial localization of several multiprotein complexes involved in U-insertion/deletion RNA editing in trypanosome mitochondria. The editing complexes are located in one or two antipodal nodes adjacent to the kinetoplast DNA (kDNA) disk, which are distinct from but associated with the minicircle catenation nodes. In some cases the proteins are in a bilateral sheet configuration. We also found that mitoribosomes have a nodal configuration. This type of organization is consistent with evidence for protein and RNA interactions of multiple editing complexes to form an ~40S editosome and also an interaction of editosomes with mitochondrial ribosomes.

New Approaches to Systematics of Trypanosomatidae: Criteria for Taxonomic (Re)description.

While dixenous trypanosomatids represent one of the most dangerous pathogens for humans and domestic animals, their monoxenous relatives have frequently become model organisms for studies of diversity of parasitic protists and host-parasite associations. Yet, the classification of the family Trypanosomatidae is not finalized and often confusing. Here we attempt to make a blueprint for future studies in this field. We would like to elicit a discussion about an updated procedure, as traditional taxonomy was not primarily designed to be used for protists, nor can molecular phylogenetics solve all the problems alone. The current status, specific cases, and examples of generalized solutions are presented under conditions where practicality is openly favored over rigid taxonomic codes or blind phylogenetic approach.

Identification of the mitochondrially encoded subunit 6 of F1FO ATPase in Trypanosoma brucei.

Kinetoplast maxicircle DNA of trypanosomatids encodes eighteen proteins. RNA editing is required to confer translatability to mRNA for twelve of these. Sequence conservation of the predicted hydrophobic polypeptides indicates that they represent functional components of the respiratory chain. Yet, so far only two of those, cytochrome c oxidase subunit I and apocytochrome b of cytochrome c reductase, have been identified with biochemical methods. Here we report on identification of A6 subunit of F1FO ATPase encoded by a pan-edited mRNA in Trypanosoma brucei. The polypeptide was present among the (35)S-labeled mitochondrial translation products characterized by anomalous migration in denaturing 2D gels. It was identified as an ATPase subunit by co-migration with this complex in Blue Native 2D gels. A partial N-terminal sequence of the corresponding polypeptide present in the gel-purified ATPase complex from Leishmania tarentolae was consistent with the predicted A6 sequence.

Kinetoplast DNA-encoded ribosomal protein S12: a possible functional link between mitochondrial RNA editing and translation in Trypanosoma brucei.

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, which are encoded by the kinetoplast genome, and more than 150 proteins encoded in the nucleus and imported from the cytoplasm. However, a single ribosomal protein RPS12 is encoded by the kinetoplast DNA (kDNA) in all trypanosomatid species examined. As typical for these organisms, the gene itself is cryptic and its transcript undergoes an extensive U-insertion/deletion editing. An evolutionary trend to reduce or eliminate RNA editing could be traced with other cryptogenes, but the invariably pan-edited RPS12 cryptogene is apparently spared. Here we inquired whether editing of RPS12 mRNA is essential for mitochondrial translation. By RNAi-mediated knockdowns of RNA editing complexes and inducible knock-in of a key editing enzyme in procyclic parasites, we could reversibly downregulate production of edited RPS12 mRNA and, by inference, synthesis of this protein. While inhibition of editing decreased edited mRNA levels, the translation of edited (Cyb) and unedited (COI) mRNAs was blocked. Furthermore, the population of SSU-related 45S complexes declined upon inactivation of editing and so did the amount of mRNA-bound ribosomes. In bloodstream parasites, which lack active electron transport chain but still require translation of ATP synthase subunit 6 mRNA (A6), both edited RPS12 and A6 mRNAs were detected in translation complexes. Collectively, our results indicate that a single ribosomal protein gene retained by the kinetoplast mitochondrion serves as a possible functional link between editing and translation processes and provide the rationale for the evolutionary conservation of RPS12 pan-editing.

The importance of the 45 S ribosomal small subunit-related complex for mitochondrial translation in Trypanosoma brucei.

The mitochondrial 45 S SSU* complex in Trypanosoma brucei contains the 9 S SSU ribosomal RNA, a set of SSU ribosomal proteins, several pentatricopeptide repeat (PPR) proteins, and proteins not typically found in ribosomes, including rhodanese domain protein (Rhod) and a 200-kDa coiled-coil protein. To investigate the function of this complex, PPR29, Rhod, 200-kDa protein, and mitochondrial ribosomal protein S17 were knocked down by RNAi in procyclic T. brucei. A growth retardation phenotype, a reduction in the amount of the 45 S SSU* complexes, and the preferential inhibition of synthesis of the cytochrome c oxidase subunit I over apocytochrome b were observed as early as day 2 postinduction of RNAi. On the contrary, the down-regulation of mitochondrial ribosomal protein L3 drastically reduced the amount of the large subunit and indiscriminately inhibited mitochondrial translation. The relative amounts of translation-competent, long poly(AU)-tailed cytochrome c oxidase subunit I and edited apocytochrome b mRNAs were selectively reduced by ablation of the 45 S SSU* complex. The formation of the 80 S translation complexes, identified by association of the long-tailed mRNAs with the mitoribosomes, was also disrupted. On the other hand, the relative amount of long-tailed edited RPS12 mRNA was not substantially affected, and there was no noticeable effect on the RPS12 translation complexes. In bloodstream trypanosomes, the amount of the 45 S complexes was drastically reduced compared with procyclics. We propose that the 45 S SSU* complex represents a factor required for normal mitochondrial translation that may have selective effects on different mRNAs.

Diversity and phylogeny of insect trypanosomatids: all that is hidden shall be revealed.

Monoxenous trypanosomatids, which are usually regarded as benign dwellers of the insect alimentary tract, represent a relatively obscure group within the family Trypanosomatidae. This field of study has long been in disarray with the genus level taxonomy of this group remaining artificial, species criteria elusive, host specificity and occurrence poorly known, and their diversity mostly unexplored. The time has arrived to remedy this situation: a phylogenetic approach has been applied to taxa recognition and description, and a culture-independent (PCR-based) approach for detection and identification of organisms in nature has made it feasible to study the diversity of the group. Although more than 100 typing units have been discovered recently, these appear to represent a small segment of trypanosomatid biodiversity, which still remains to be uncovered.

New species of insect trypanosomatids from Costa Rica and the proposal for a new subfamily within the Trypanosomatidae.

Several new species of trypanosomatids (Euglenozoa, Kinetoplastea, Trypanosomatidae), isolated from the intestines of Neotropical insects (Heteroptera), were genotyped on the basis of spliced leader RNA, and also defined phylogenetically using gene sequences of small subunit ribosomal RNA and glycosomal glyceraldehyde phosphate dehydrogenase. The taxonomic descriptions also included characterization using morphometry and electron microscopy. Our phylogenetic analyses placed the new species within the clade, previously designated "SE" for "Slowly Evolving" sequences of ribosomal RNA genes, a clade that also includes numerous monoxenous parasites of insects from the genera Crithidia, Leptomonas, and Wallaceina, as well as the dixenous genus Leishmania. Based on the high phylogenetic support for this clade, which is consistently recovered in all recent phylogenetic reconstructions, a proposal is put forward to recognize this natural taxon as a new subfamily, Leishmaniinae, within the family Trypanosomatidae.

Microbotryozyma collariae gen. nov., sp. nov., a basidiomycetous yeast isolated from a plant bug Collaria oleosa (Miridae).

Two strains of a basidiomycetous yeast were derived from an insect trypanosomatid culture isolated from the intestine of a plant bug, Collaria oleosa (Heteroptera: Miridae), collected in Costa Rica. The yeast did not form ballistoconidia but reproduced only by budding. Teliospores were not observed in individual and crossed cultures of each strain. Morphological and other taxonomic characteristics of the yeast were similar to those of the species in the polyphyletic genus Rhodotorula. However, molecular phylogeny inferred from the internal transcribed spacers and D1/D2 region of the large subunit rRNA gene showed that the strains represent a new species placed among the smut fungi in the family Ustilentylomataceae, which includes Aurantiosporium subnitens, Fulvisporium restifaciens, Ustilentyloma fluitans, and Rhodotorula hordea. Given the well distinguished phylogenetic position of this novel species within the Ustilentylomataceae, we propose Microbotryozyma collariae gen. nov., sp. nov. to accommodate the yeast isolated from C. oleosa, with strain American Type Culture Collection MYA-4666(T) (= PRA303-1S = CBS 12537) designated as the type strain.

Cosmopolitan distribution of a trypanosomatid Leptomonas pyrrhocoris.

A trypanosomatid species, designated as Typing Unit 1 (TU1) by sequences of SL RNA gene repeats, has been found in the intestine of pyrrhocorids (Insecta: Heteroptera) in Europe, Mediterranean, Central America and some parts of Asia and Africa. Phylogenetic analysis of the SL repeat sequences has shown that the isolates group in the tree according to their geographic origin. The maximal sequence divergence was observed in parasites from Neotropics suggesting the origin within and subsequent migrations from this region. The global distribution of the parasite could have been facilitated by ubiquity of its hosts that include several genera of the family Pyrrhocoridae. In Europe the TU1 flagellates frequently occur in Pyrrhocoris apterus, the host of Leptomonas pyrrhocorisZotta, 1912, a species that had been insufficiently defined by host and light microscopy level morphology. Herein, the Zotta's species description has been amended to include the TU1 SL RNA repeat, SSU rRNA, glycosomal GAPDH gene sequences, as well as ultrastructure. In addition, Leptomonas scantii n. sp. with an overlapping host range has been described. Moreover, 10 typing units of trypanosomatids found in the pyrrhocorid hosts demonstrate the extent of variability of trypanosomatids occurring in one host family.