First finding of free-living representatives of Prokinetoplastina and their nuclear and mitochondrial genomes.

Kinetoplastids are heterotrophic flagellated protists, including important parasites of humans and animals (trypanosomatids), and ecologically important free-living bacterial consumers (bodonids). Phylogenies have shown that the earliest-branching kinetoplastids are all parasites or obligate endosymbionts, whose highly-derived state makes reconstructing the ancestral state of the group challenging. We have isolated new strains of unusual free-living flagellates that molecular phylogeny shows to be most closely related to endosymbiotic and parasitic Perkinsela and Ichthyobodo species that, together with unidentified environmental sequences, form the clade at the base of kinetoplastids. These strains are therefore the first described free-living prokinetoplastids, and potentially very informative in understanding the evolution and ancestral states of morphological and molecular characteristics described in other kinetoplastids. Overall, we find that these organisms morphologically and ultrastructurally resemble some free-living bodonids and diplonemids, and possess nuclear genomes with few introns, polycistronic mRNA expression, high coding density, and derived traits shared with other kinetoplastids. Their genetic repertoires are more diverse than the best-studied free-living kinetoplastids, which is likely a reflection of their higher metabolic potential. Mitochondrial RNAs of these new species undergo the most extensive U insertion/deletion editing reported so far, and limited deaminative C-to-U and A-to-I editing, but we find no evidence for mitochondrial trans-splicing.

Glycosome heterogeneity in kinetoplastids.

Kinetoplastid parasites have essential organelles called glycosomes that are analogous to peroxisomes present in other eukaryotes. While many of the processes that regulate glycosomes are conserved, there are several unique aspects of their biology that are divergent from other systems and may be leveraged as therapeutic targets for the treatment of kinetoplastid diseases. Glycosomes are heterogeneous organelles that likely exist as sub-populations with different protein composition and function in a given cell, between individual cells, and between species. However, the limitations posed by the small size of these organelles makes the study of this heterogeneity difficult. Recent advances in the analysis of small vesicles by flow-cytometry provide an opportunity to overcome these limitations. In this review, we describe studies that document the diverse nature of glycosomes and propose an approach to using flow cytometry and organelle sorting to study the diverse composition and function of these organelles. Because the cellular machinery that regulates glycosome protein import and biogenesis is likely to contribute, at least in part, to glycosome heterogeneity we highlight some ways in which the glycosome protein import machinery differs from that of peroxisomes in other eukaryotes.

Maxi-circles, glycosomes, gene transposition, expression sites, transsplicing, transferrin receptors and base J.

This is a personal story of the author of his research on trypanosomatids, covering a period of 1970-2015. Some of the highlights include the discovery of new aspects of kDNA, the mini-circle heterogeneity and the maxi-circle; the glycosome; the discovery of gene transposition as a major mechanism for antigenic variation; trans-splicing as an essential step in the synthesis of all trypanosome mRNAs; Pulsed Field Gradient gels to size-fractionate chromosome-sized DNA molecules of protozoa; the sequence of trypanosome telomeres and their growth and contraction; the first ABC-transporter of trypanosomatids, LtpgpA; the variable transferrin receptor of T. brucei and its role in Fe uptake; and base J, its structure, biosynthesis and function.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era.

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas.

BACKGROUND: Leptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins. RESULTS: During change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (ß-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the ß-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes. CONCLUSION: Our study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information presented in our present work has importance for drug design targeted to kill L. donovani in humans but not affect the human form of the prenylation enzymes.

Developmental regulation of gene expression in the absence of transcriptional control: the case of kinetoplastids.

Kinetoplastids, including the human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania, are the only known organisms that do not regulate the transcription of protein coding genes transcribed by RNA polymerase II. Yet, profound changes in gene expression are induced by many different external stimuli and stresses, the extreme example are cascades of changes in gene expression initiated by differentiation triggers that ultimately and irreversibly result in the massive morphological and metabolic changes observed during life-cycle progression. This review explores how kinetoplastids change gene expression by looking at life-cycle stage specific changes in chromatin, mRNA processing, mRNA stability, mRNA translation, protein stability and protein modifications.

Ichthyobodo salmonis sp. n. (Ichthyobodonidae, Kinetoplastida), an euryhaline ectoparasite infecting Atlantic salmon (Salmo salar L.).

Phylogenetic analyses of SSU rDNA sequences have previously revealed the existence of 2 Ichthyobodo species able to infect Atlantic salmon (Salmo salar L.). Ichthyobodo necator sensu stricto (s.s.) is assumed to be a freshwater parasite, while a genetically distinct but undescribed species, Ichthyobodo sp. II sensu Todal et al. (2004) have been detected on Atlantic salmon in both fresh- and seawater. In the present study a morphological description of Ichthyobodo sp. II from the gills of salmon reared in fresh-, brackish- and seawater is presented, using both light- and electron microscopy. Comparative morphometry show that Ichthyobodo sp. II from both freshwater and seawater displays a different cell shape, and is significantly smaller than I. necator s.s. Also, ultrastructural characteristics distinguish these two species, notably differences in the attachment region and the presence of spine-like surface projections in Ichthyobodo sp. II. Based on both unique SSU rDNA sequences and morphological characteristics, we conclude that Ichthyobodo sp. II. represents a novel species for which we propose the name Ichthyobodo salmonis sp. n.

Caracterização morfológica, bioquímica e molecular de vickermania itaguaiensis n gen, n sp (protozoa, kinetoplastida) / Morphological, biochemical and molecular vickerman itaguaiensis n o gen, n sp (Protozoa, Kinetoplastida)

A família Trypanosomatidae inclui parasitas de uma grande variedade de vertebrados, invertebrados (principalmente insetos), plantas e algumas espécies de protozoários, sendo, depois do nematóides, os de maior distribuição de hospedeiros na natureza. No presente trabalho, um novo isolado foi obtido por coprocultivo de trato intestinal de Leptoglossus stigma Herb, 1784 (Hemíptera, Coreidae), capturado no município de Itaguaí/RJ. O isolado original e três clones (obtidos por citometria de fluxo) foram depositados na “Coleção de Flagelados do Laboratório de Transmissores de Leishmanioses.” Um clone foi caracterizado por diversas abordagens em comparação com espécies de referência de diferentes gêneros. Foi analisado o crescimento em meio de cultivo em intervalos de 24 h, entre 48-144 h, a diferenciação celular e a morfometria dos principais estágios evolutivos encontrados. A análise de isoenzimas foi realizada utilizando-se os seguintes sistemas: GPI, PGM, 6PGDH, HK, ACON, MPI, FUM, IDH, MDH e ACON. RAPD-PCR foi realizado utilizando-se 6 iniciadores, conjuntamente com outros tripanosomatídeos. Os dados destas análises foram processados numericamente e submetidos à análise computacional utilizando-se o coeficiente de associação Jaccard e o SSU rRNA e o fragmento analisado foi alinhado com vinte e uma seqüências depositadas no GenBank, gerando uma árvore filogenética resultante do pareamento. O conjunto de resultados deste trabalho sugere que a amostra obtida constitui nova espécie, pertencendo a um novo gênero, nomeada Vickermania itaguaiensis.

Topoisomerases of kinetoplastid parasites: why so fascinating?

DNA topoisomerases are the key enzymes involved in carrying out high precision DNA transactions inside the cells. However, they are detrimental to the cell when a wide variety of topoisomerase-targeted drugs generate cytotoxic lesions by trapping the enzymes in covalent complexes on the DNA. The discovery of unusual heterodimeric topoisomerase I in kinetoplastid family added a new twist in topoisomerase research related to evolution, functional conservation and their preferential sensitivity to Camptothecin. On the other hand, structural and mechanistic studies on kinetoplastid topoisomerase II delineate some distinguishing features that differentiate the parasitic enzyme from its prokaryotic and eukaryotic counterparts. This review summarizes the recent advances in research in kinetoplastid topoisomerases, their evolutionary significance and the death of the unicellular parasite Leishmania donovani induced by topoisomerase I inhibitor camptothecin.

Basal body and flagellum mutants reveal a rotational constraint of the central pair microtubules in the axonemes of trypanosomes.

Productive beating of eukaryotic flagella and cilia requires a strict regulation of axonemal dynein activation. Fundamental to any description of axonemal beating is an understanding of the significance of the central pair microtubules and the degree to which central pair rotation has a role. However, for the majority of organisms, it is unclear whether the central pair actually rotates. Using an extra-axonemal structure as a fixed reference, we analysed the orientation of the central pair in African trypanosomes and other kinetoplastid protozoa. A geometric correction allowed the superposition of data from many cross-sections, demonstrating that the axis of the central pair is invariant and that there is no central pair rotation in these organisms. Analysis of mutants depleted in particular flagellar and basal body proteins [gamma-tubulin, delta-tubulin, Parkin co-regulated gene product (PACRG) or the paraflagellar rod protein PFR2] allowed a dissection of the mechanisms for central pair constraint. This demonstrated that orientation is independent of flagellum attachment and beating, but is influenced by constraints along its length and is entirely dependent on correct positioning at the basal plate.

Mitochondrial differentiation in kinetoplastid protozoa: a plethora of RNA controls.

Differentiation of kinetoplastid protozoa during their complex life cycles is accompanied by stepwise changes in mitochondrial functions. Recent studies have begun to reveal multilevel post-transcriptional regulatory mechanisms by which the expression of the nuclear and mitochondrially encoded components of respiratory enzymes is coordinated, as well as the identities of some general and gene-specific factors controlling mitochondrial differentiation.

The endocytic apparatus of the kinetoplastida. Part II: machinery and components of the system.

Endocytic systems within eukaryotic cells are a diverse set of intracellular transport pathways responsible for uptake, recycling, interaction with the exocytic system and degradation of molecules. Each of these pathways requires the interaction of distinct protein components that function in macromolecule sorting, control of transport rates and in membrane biogenesis. In the second of two articles on kinetoplastida endocytosis, the endocytic system in Trypanosoma brucei is considered as a model, and the molecules that control this system and the protein components of the endocytic pathway are discussed. We also consider novel mechanisms for sorting that have been proposed to operate in trypanosomes.

Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii.

A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms. Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution. Significant progress in studying the cell biology of this parasite has been accomplished over the last few years. Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T. gondii. In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.

Comparative morphology of kinetoplastids.

The common structural plan of bodonids, cryptobiids and trypanosomatids permits placing them in one phylum Kinetoplastidae. Analysis of evolutionary changes in the initial "bodonid" structural plan in separate groups within the Kinetoplastidae, illustrated by an evolutionary series of homologous characters, enables us to conclude that the cryptobiids occupy an intermediate position in kinetoplastid evolution between the free-living bodonids and Trypanosoma spp from lower vertebrates and can be considered to be potential ancestors of the latter.

Kinetoplastidae display naturally occurring ancillary DNA-containing structures.

Kinetoplast-derived, DNA-containing structures were found in several members of the order Kinetoplastida. The structures, for which we propose the name ancillary DNA-containing structures (aDNA), were discovered during the course of low-light-level video fluorescence microscopy studies using several nucleic acid-specific fluorescent reagents. DNase treatment and supravital stain with Höechst 33342 confirmed that aDNA is not an artifact of specimen preparation. Fluorescent in situ hybridization using either a 122-bp kinetoplast DNA-specific probe derived from a conserved region of minicircle DNA or a 188-bp nuclear DNA-specific probe derived from highly repetitive nuclear DNA demonstrated that aDNA is derived from the kinetoplast and not the nucleus. However, the structures do not contain minicircle DNA replication intermediates. Immunofluorescence assays using an anti-mitochondrial protein antibody, anti-mtp70, demonstrated that the structures contain mitochondrial protein and confirmed their kinetoplast origin. The frequency of occurrence of aDNA varies markedly between members of the Kinetoplastida. In the case of Trypanosoma cruzi stocks, the percentage of cells with aDNA was positively correlated to the population doubling time of the stock. However, there is no statistically significant relationship between the developmental or replicative stage of the parasite and the frequency of aDNA. An inhibitor of DNA topoisomerase I had no effect upon the frequency of aDNA. An inhibitor of DNA topoisomerase II gave equivocal results depending upon the parasite stock used. We speculate that aDNA may be the morphological consequence of a yet-to-be-determined biological process intrinsic to but variable within the Kinetoplastida.