Genome Analysis of Endotrypanum and Porcisia spp., Closest Phylogenetic Relatives of Leishmania, Highlights the Role of Amastins in Shaping Pathogenicity.

While numerous genomes of Leishmania spp. have been sequenced and analyzed, an understanding of the evolutionary history of these organisms remains limited due to the unavailability of the sequence data for their closest known relatives, Endotrypanum and Porcisia spp., infecting sloths and porcupines. We have sequenced and analyzed genomes of three members of this clade in order to fill this gap. Their comparative analyses revealed only minute differences from Leishmaniamajor genome in terms of metabolic capacities. We also documented that the number of genes under positive selection on the Endotrypanum/Porcisia branch is rather small, with the flagellum-related group of genes being over-represented. Most significantly, the analysis of gene family evolution revealed a substantially reduced repertoire of surface proteins, such as amastins and biopterin transporters BT1 in the Endotrypanum/Porcisia species when compared to amastigote-dwelling Leishmania. This reduction was especially pronounced for δ-amastins, a subfamily of cell surface proteins crucial in the propagation of Leishmania amastigotes inside vertebrate macrophages and, apparently, dispensable for Endotrypanum/Porcisia, which do not infect such cells.

Characterization of a new cosmopolitan genus of trypanosomatid parasites, Obscuromonas gen. nov. (Blastocrithidiinae subfam. nov.).

The expanding phylogenetic tree of trypanosomatid flagellates (Kinetoplastea: Trypanosomatidae) contains a long-known and phylogenetically well-supported species-rich lineage that was provisionally named as the 'jaculum' clade. Its members were found in representatives of several unrelated families of heteropteran bugs captured in South and Central America, Europe, Africa, and Asia. However, this group resisted introduction into the culture, a needed prerequisite for its proper characterization. Here we describe four new cultivable species, which parasitize various parts of their hosts' intestine, including the thoracic and abdominal part of the midgut, hindgut, and Malpighian tubules. Morphologically, the cultured flagellates vary from relatively short stumpy promastigotes to long slender leptomonad cells. Some species form straphangers (cyst-like amastigotes) both in vivo and in vitro, initially attached to the basal part of the flagellum of the mother cell, from which they subsequently detach. To formally classify this enigmatic monophyletic cosmopolitan clade, we erected Obscuromonas gen. nov., including five species: O. modryi sp. nov. (isolated from the true bug host species Riptortus linearis captured in the Philippines), O. volfi sp. nov. (from Catorhintha selector, Curaçao), O. eliasi sp. nov. (from Graptostethus servus, Papua New Guinea), O. oborniki sp. nov. (from Aspilocoryphus unimaculatus, Madagascar), and O. jaculum comb. nov. (from Nepa cinerea, France). Obscuromonas along with the genus Blastocrithidia belongs to the newly established Blastocrithidiinae subfam. nov.

Universal and taxon-specific trends in protein sequences as a function of age.

Extant protein-coding sequences span a huge range of ages, from those that emerged only recently to those present in the last universal common ancestor. Because evolution has had less time to act on young sequences, there might be 'phylostratigraphy' trends in any properties that evolve slowly with age. A long-term reduction in hydrophobicity and hydrophobic clustering was found in previous, taxonomically restricted studies. Here we perform integrated phylostratigraphy across 435 fully sequenced species, using sensitive HMM methods to detect protein domain homology. We find that the reduction in hydrophobic clustering is universal across lineages. However, only young animal domains have a tendency to have higher structural disorder. Among ancient domains, trends in amino acid composition reflect the order of recruitment into the genetic code, suggesting that the composition of the contemporary descendants of ancient sequences reflects amino acid availability during the earliest stages of life, when these sequences first emerged.

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach.

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

Vickermania gen. nov., trypanosomatids that use two joined flagella to resist midgut peristaltic flow within the fly host.

BACKGROUND: The family Trypanosomatidae encompasses parasitic flagellates, some of which cause serious vector-transmitted diseases of humans and domestic animals. However, insect-restricted parasites represent the ancestral and most diverse group within the family. They display a range of unusual features and their study can provide insights into the biology of human pathogens. Here we describe Vickermania, a new genus of fly midgut-dwelling parasites that bear two flagella in contrast to other trypanosomatids, which are unambiguously uniflagellate. RESULTS: Vickermania has an odd cell cycle, in which shortly after the division the uniflagellate cell starts growing a new flagellum attached to the old one and preserves their contact until the late cytokinesis. The flagella connect to each other throughout their whole length and carry a peculiar seizing structure with a paddle-like apex and two lateral extensions at their tip. In contrast to typical trypanosomatids, which attach to the insect host's intestinal wall, Vickermania is separated from it by a continuous peritrophic membrane and resides freely in the fly midgut lumen. CONCLUSIONS: We propose that Vickermania developed a survival strategy that relies on constant movement preventing discharge from the host gut due to intestinal peristalsis. Since these parasites cannot attach to the midgut wall, they were forced to shorten the period of impaired motility when two separate flagella in dividing cells interfere with each other. The connection between the flagella ensures their coordinate movement until the separation of the daughter cells. We propose that Trypanosoma brucei, a severe human pathogen, during its development in the tsetse fly midgut faces the same conditions and follows the same strategy as Vickermania by employing an analogous adaptation, the flagellar connector.

Ultraconserved element bait set for trypanosomatida target enrichment and phylogenetics.

Lack of knowledge of taxonomic biodiversity and reliable genetic markers in Trypanosomatidae limit our understanding of their phylogenetic relationships. Ultraconserved elements (UCEs) have improved phylogenetic analyses and inferences in many vertebrate and invertebrate taxa. However, it is unknown whether protozoans have these markers, their abundance, and if these could be reliably used for phylogenetics. In this study I design a target enrichment bait set for UCE loci for this group. In silico testing showed good loci recovery rates across 63 taxa and produced consistent, highly supported phylogenetic trees. This bait set adds a new resource of useful genetic markers for Trypanosomatidae phylogenetics.

Analysing ambiguities in trypanosomatids taxonomy by barcoding.

BACKGROUND: Biodiversity screens and phylogenetic studies are dependent on reliable DNA sequences in public databases. Biological collections possess vouchered specimens with a traceable history. Therefore, DNA sequencing of samples available at institutional collections can greatly contribute to taxonomy, and studies on evolution and biodiversity. METHODS: We sequenced part of the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and the SSU rRNA (V7/V8) genes from 102 trypanosomatid cultures, which are available on request at www.colprot.fiocruz.br. OBJECTIVE: The main objective of this work was to use phylogenetic inferences, using the obtained DNA sequences and those from representatives of all Trypanosomatidae genera, to generate phylogenetic trees that can simplify new isolates screenings. FINDINGS: A DNA sequence is provided for the first time for several isolates, the phylogenetic analysis allowed the classification or reclassification of several specimens, identification of candidates for new genera and species, as well as the taxonomic validation of several deposits. MAIN CONCLUSIONS: This survey aimed at presenting a list of validated species and their associated DNA sequences combined with a short historical overview of each isolate, which can support taxonomic and biodiversity research and promote culture collections.

A novel endosymbiont-containing trypanosomatid Phytomonas borealis sp. n. from the predatory bug Picromerus bidens (Heteroptera: Pentatomidae).

Here we describe the new trypanosomatid, Phytomonas borealis sp. n., from the midgut of the spiked shieldbugs, Picromerus bidens (Linnaeus), collected in two locations, Novgorod and Pskov Oblasts of Russia. The phylogenetic analyses, based on the 18S rRNA gene, demonstrated that this flagellate is a sister species to the secondary monoxenous Phytomonas nordicus Frolov et Malysheva, 1993, which was concurrently documented in the same host species in Pskov Oblast. Unlike P. nordicus, which can complete its development (including exit to haemolymph and penetration into salivary glands) in Picromerus bidens, the new species did not form any extraintestinal stages in the host. It also did not produce endomastigotes, indispensable for transmission in other Phytomonas spp. These observations, along with the fact that P. bidens overwinters at the egg stage, led us to the conclusion that the examined infections with P. borealis were non-specific. Strikingly, the flagellates from the Novgorod population contained prokaryotic endosymbionts, whereas the parasites from the second locality were endosymbiont-free. This is a first case documenting presence of intracellular symbiotic bacteria in Phytomonas spp. We suggest that this novel endosymbiotic association arose very recently and did not become obligate yet. Further investigation of P. borealis and its intracellular bacteria may shed light on the origin and early evolution of endosymbiosis in trypanosomatids.

The complexity and diversity of the actin cytoskeleton of trypanosomatids.

Trypanosomatids are a monophyletic group of parasitic flagellated protists belonging to the order Kinetoplastida. Their cytoskeleton is primarily made up of microtubules in which no actin microfilaments have been detected. Although all these parasites contain actin, it is widely thought that their actin cytoskeleton is reduced when compared to most eukaryotic organisms. However, there is increasing evidence that it is more complex than previously thought. As in other eukaryotic organisms, trypanosomatids encode for a conventional actin that is expected to form microfilament-like structures, and for members of three conserved actin-related proteins probably involved in microfilament nucleation (ARP2, ARP3) and in gene expression regulation (ARP6). In addition to these canonical proteins, also encode for an expanded set of actins and actin-like proteins that seem to be restricted to kinetoplastids. Analysis of their amino acid sequences demonstrated that, although very diverse in primary sequence when compared to actins of model organisms, modelling of their tertiary structure predicted the presence of the actin fold in all of them. Experimental characterization has been done for only a few of the trypanosomatid actins and actin-binding proteins. The most studied is the conventional actin of Leishmania donovani (LdAct), which unusually requires both ATP and Mg2+ for polymerization, unlike other conventional actins that do not require ATP. Additionally, polymerized LdAct tends to assemble in bundles rather than in single filaments. Regulation of actin polymerization depends on their interaction with actin-binding proteins. In trypanosomatids, there is a reduced but sufficient core of actin-binding proteins to promote microfilament nucleation, turnover and stabilization. There are also genes encoding for members of two families of myosin motor proteins, including one lineage-specific. Homologues to all identified actin-family proteins and actin-binding proteins of trypanosomatids are also present in Paratrypanosoma confusum (an early branching trypanosomatid) and in Bodo saltans (a closely related free-living organism belonging to the trypanosomatid sister order of Bodonida) suggesting they were all present in their common ancestor. Secondary losses of these genes may have occurred during speciation within the trypanosomatids, with salivarian trypanosomes having lost many of them and stercorarian trypanosomes retaining most.

A review of the systematics, species identification and diagnostics of the Trypanosomatidae using the maxicircle kinetoplast DNA: from past to present.

The Trypanosomatid family are a diverse and widespread group of protozoan parasites that belong to the higher order class Kinetoplastida. Containing predominantly monoxenous species (i.e. those having only a single host) that are confined to invertebrate hosts, this class is primarily known for its pathogenic dixenous species (i.e. those that have two hosts), serving as the aetiological agents of the important neglected tropical diseases including leishmaniasis, American trypanosomiasis (Chagas disease) and human African trypanosomiasis. Over the past few decades, a multitude of studies have investigated the diversity, classification and evolutionary history of the trypanosomatid family using different approaches and molecular targets. The mitochondrial-like DNA of the trypanosomatid parasites, also known as the kinetoplast, has emerged as a unique taxonomic and diagnostic target for exploring the evolution of this diverse group of parasitic eukaryotes. This review discusses recent advancements and important developments that have made a significant impact in the field of trypanosomatid systematics and diagnostics in recent years.

Proteins involved in the biosynthesis of lipophosphoglycan in Leishmania: a comparative genomic and evolutionary analysis.

BACKGROUND: Leishmania spp. are digenetic parasites capable of infecting humans and causing a range of diseases collectively known as leishmaniasis. The main mechanisms involved in the development and permanence of this pathology are linked to evasion of the immune response. Crosstalk between the immune system and particularities of each pathogenic species is associated with diverse disease manifestations. Lipophosphoglycan (LPG), one of the most important molecules present on the surface of Leishmania parasites, is divided into four regions with high molecular variability. Although LPG plays an important role in host-pathogen and vector-parasite interactions, the distribution and phylogenetic relatedness of the genes responsible for its synthesis remain poorly explored. The recent availability of full genomes and transcriptomes of Leishmania parasites offers an opportunity to leverage insight on how LPG-related genes are distributed and expressed by these pathogens. RESULTS: Using a phylogenomics-based framework, we identified a catalog of genes involved in LPG biosynthesis across 22 species of Leishmania from the subgenera Viannia and Leishmania, as well as 5 non-Leishmania trypanosomatids. The evolutionary relationships of these genes across species were also evaluated. Nine genes related to the production of the glycosylphosphatidylinositol (GPI)-anchor were highly conserved among compared species, whereas 22 genes related to the synthesis of the repeat unit presented variable conservation. Extensive gain/loss events were verified, particularly in genes SCG1-4 and SCA1-2. These genes act, respectively, on the synthesis of the side chain attached to phosphoglycans and in the transfer of arabinose residues. Phylogenetic analyses disclosed evolutionary patterns reflective of differences in host specialization, geographic origin and disease manifestation. CONCLUSIONS: The multiple gene gain/loss events identified by genomic data mining help to explain some of the observed intra- and interspecies variation in LPG structure. Collectively, our results provide a comprehensive catalog that details how LPG-related genes evolved in the Leishmania parasite specialization process.

Analysing ambiguities in trypanosomatids taxonomy by barcoding

BACKGROUND Biodiversity screens and phylogenetic studies are dependent on reliable DNA sequences in public databases. Biological collections possess vouchered specimens with a traceable history. Therefore, DNA sequencing of samples available at institutional collections can greatly contribute to taxonomy, and studies on evolution and biodiversity. METHODS We sequenced part of the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and the SSU rRNA (V7/V8) genes from 102 trypanosomatid cultures, which are available on request at www.colprot.fiocruz.br. OBJECTIVE The main objective of this work was to use phylogenetic inferences, using the obtained DNA sequences and those from representatives of all Trypanosomatidae genera, to generate phylogenetic trees that can simplify new isolates screenings. FINDINGS A DNA sequence is provided for the first time for several isolates, the phylogenetic analysis allowed the classification or reclassification of several specimens, identification of candidates for new genera and species, as well as the taxonomic validation of several deposits. MAIN CONCLUSIONS This survey aimed at presenting a list of validated species and their associated DNA sequences combined with a short historical overview of each isolate, which can support taxonomic and biodiversity research and promote culture collections.

Insect trypanosomatids in Papua New Guinea: high endemism and diversity.

The extreme biological diversity of Oceanian archipelagos has long stimulated research in ecology and evolution. However, parasitic protists in this geographic area remained neglected and no molecular analyses have been carried out to understand the evolutionary patterns and relationships with their hosts. Papua New Guinea (PNG) is a biodiversity hotspot containing over 5% of the world's biodiversity in less than 0.5% of the total land area. In the current work, we examined insect heteropteran hosts collected in PNG for the presence of trypanosomatid parasites. The diversity of insect flagellates was analysed, to our knowledge for the first time, east of Wallace's Line, one of the most distinct biogeographic boundaries of the world. Out of 907 investigated specimens from 138 species and 23 families of the true bugs collected in eight localities, 135 (15%) were infected by at least one trypanosomatid species. High species diversity of captured hosts correlated with high diversity of detected trypanosomatids. Of 46 trypanosomatid Typing Units documented in PNG, only eight were known from other geographic locations, while 38 TUs (~83%) have not been previously encountered. The widespread trypanosomatid TUs were found in both widely distributed and endemic/sub-endemic insects. Approximately one-third of the endemic trypanosomatid TUs were found in widely distributed hosts, while the remaining species were confined to endemic and sub-endemic insects. The TUs from PNG form clades with conspicuous host-parasite coevolutionary patterns, as well as those with a remarkable lack of this trait. In addition, our analysis revealed new members of the subfamilies Leishmaniinae and Strigomonadinae, potentially representing new genera of trypanosomatids.

Non-Leishmania Parasite in Fatal Visceral Leishmaniasis-Like Disease, Brazil.

Through whole-genome sequencing analysis, we identified non-Leishmania parasites isolated from a man with a fatal visceral leishmaniasis-like illness in Brazil. The parasites infected mice and reproduced the patient's clinical manifestations. Molecular epidemiologic studies are needed to ascertain whether a new infectious disease is emerging that can be confused with leishmaniasis.

An enigmatic catalase of Blastocrithidia.

Here we report that trypanosomatid flagellates of the genus Blastocrithidia possess catalase. This enzyme is not phylogenetically related to the previously characterized catalases in other monoxenous trypanosomatids, suggesting that their genes have been acquired independently. Surprisingly, Blastocrithidia catalase is less enzymatically active, compared to its counterpart from Leptomonas pyrrhocoris, posing an intriguing biological question why this gene has been retained in the evolution of trypanosomatids.

Comparative molecular cell biology of phototrophic euglenids and parasitic trypanosomatids sheds light on the ancestor of Euglenozoa.

Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual 'J' base (ß-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear.

Development of Phytomonas lipae sp. n. (Kinetoplastea: Trypanosomatidae) in the true bug Coreus marginatus (Heteroptera: Coreidae) and insights into the evolution of life cycles in the genus Phytomonas.

Here we described a new trypanosomatid species, Phytomonas lipae, parasitizing the dock bug Coreus marginatus based on axenic culture and in vivo material. Using light and electron microscopy we characterized the development of this flagellate in the intestine, hemolymph and salivary glands of its insect host. The intestinal promastigotes of Phytomonas lipae do not divide and occur only in the anterior part of the midgut. From there they pass into hemolymph, increasing in size, and then to salivary glands, where they actively proliferate without attachment to the host's epithelium and form infective endomastigotes. We conducted molecular phylogenetic analyses based on 18s rRNA, gGAPDH and HSP83 gene sequences, of which the third marker performed the best in terms of resolving phylogenetic relationships within the genus Phytomonas. Our inference demonstrated rather early origin of the lineage comprising the new species, right after that of P. oxycareni, which represents the earliest known branch within the Phytomonas clade. This allowed us to compare the development of P. lipae and three other Phytomonas spp. in their insect hosts and reconstruct the vectorial part of the life cycle of their common ancestor.

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.

High Prevalence and Endemism of Trypanosomatids on a Small Caribbean Island.

We describe the monoxenous trypanosomatids parasitizing true bugs and flies on the island of Curaçao. Out of 248 examined true bugs belonging to 17 species, 93 individuals were found to be infected (overall 38% prevalence) by at least one trypanosomatid species (referred to as typing units; TUs). Out of 80 flies, six were infected. All detected trypanosomatids were compared based on their 18S rRNA sequences with TUs parasitizing bugs and flies described from mainland South America, allowing us to assess their diversity and distribution. Besides Leptomonas pyrrhocoris and Leptomonas seymouri, two known species of the subfamily Leishmaniinae, our analysis revealed six new TUs falling into the groups 'jaculum', Blastocrithidia and Herpetomonas. Moreover, two new members of the genus Phytomonas and three new TUs belonging to the monophyletic group designated as 'new clade II' sensu Mol. Phylogenet. Evol, 69, 255 (2013) were isolated. The detected trypanosomatids were characterized by moderate diversity (13 TUs) species richness. Out of nine and four TUs from the heteropteran and dipteran hosts, respectively, 11 TUs have not been encountered before. Although a sampling bias may partially affect the comparison between trypanosomatid communities on Curaçao and the mainland, the high proportion of unique TUs from the former location suggests that the prominent role of islands in increasing the global diversity of macroscopic organisms may also extend to their protistan parasites.

Evolutionary origins and specialisation of membrane transport.

From unicellular protists to the largest megafauna and flora, all eukaryotes depend upon the organelles and processes of the intracellular membrane trafficking system. Well-defined machinery selectively packages and delivers material between endomembrane organelles and imports and exports material from the cell surface. This process underlies intracellular compartmentalization and facilitates myriad processes that define eukaryotic biology. Membrane trafficking is a landmark in the origins of the eukaryotic cell and recent work has begun to unravel how the revolution in cellular structure occurred.