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.

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.

Electron Microscopy Techniques Applied to Symbiont-Harboring Trypanosomatids: The Association of the Bacterium with Host Organelles.

In this chapter we describe different electron microscopy techniques such as freeze fracture, deep etching, and three-dimensional reconstruction, obtained by electron tomography or focused ion beam scanning electron microscopy (FIB-SEM), combined with quick-freezing methods in order to reveal aspects of the cell structure in trypanosomatids. For this purpose, we chose protists that evolve in a mutualistic way with a symbiotic bacterium. Such cells represent excellent models to study the positioning and distribution of organelles, since the symbiotic bacterium interacts with different organelles of the host trypanosomatid. We demonstrate that the employment of such techniques can show the proximity and even the interaction of the symbiotic bacterium with different structures of the protist host, such as the nucleus and the glycosomes. In addition, the quick-freezing approach can reveal new aspects of the gram-negative bacterial envelope, such as the presence of a greatly reduced cell wall between the two membrane units.

Airyscan Superresolution Microscopy to Study Trypanosomatid Cell Biology.

The recent introduction by Carl Zeiss Ltd. of the Airyscan detector module for their LSM880 confocal laser-scanning microscope has enabled routine superresolution microscopy to be combined with the advantages of confocal-based fluorescence imaging. Resulting enhanced spatial resolution in X, Y, and Z provides tractable opportunity to derive new insight into protein localization(s), organelle dynamics, and thence protein function within trypanosomatids or other organisms. Here, we describe methods for preparing slides, cells, and basic microscope setup for fluorescence imaging of trypanosomatids using the LSM-880 with Airyscan platform.

More than Microtubules: The Structure and Function of the Subpellicular Array in Trypanosomatids.

The subpellicular microtubule array defines the wide range of cellular morphologies found in parasitic kinetoplastids (trypanosomatids). Morphological studies have characterized array organization, but little progress has been made towards identifying the molecular mechanisms that are responsible for array differentiation during the trypanosomatid life cycle, or the apparent stability and longevity of array microtubules. In this review, we outline what is known about the structure and biogenesis of the array, with emphasis on Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, which cause life-threatening diseases in humans and livestock. We highlight unanswered questions about this remarkable cellular structure that merit new consideration in light of our recently improved understanding of how the 'tubulin code' influences microtubule dynamics to generate complex cellular structures.

Evolutionary divergent PEX3 is essential for glycosome biogenesis and survival of trypanosomatid parasites.

Trypanosomatid parasites cause devastating African sleeping sickness, Chagas disease, and Leishmaniasis that affect about 18 million people worldwide. Recently, we showed that the biogenesis of glycosomes could be the "Achilles' heel" of trypanosomatids suitable for the development of new therapies against trypanosomiases. This was shown for inhibitors of the import machinery of matrix proteins, while the distinct machinery for the topogenesis of glycosomal membrane proteins evaded investigation due to the lack of a druggable interface. Here we report on the identification of the highly divergent trypanosomal PEX3, a central component of the transport machinery of peroxisomal membrane proteins and the master regulator of peroxisome biogenesis. The trypanosomatid PEX3 shows very low degree of conservation and its identification was made possible by a combinatory approach identifying of PEX19-interacting proteins and secondary structure homology screening. The trypanosomal PEX3 localizes to glycosomes and directly interacts with the membrane protein import receptor PEX19. RNAi-studies revealed that the PEX3 is essential and that its depletion results in mislocalization of glycosomal proteins to the cytosol and a severe growth defect. Comparison of the parasites and human PEX3-PEX19 interface disclosed differences that might be accessible for drug development. The absolute requirement for biogenesis of glycosomes and its structural distinction from its human counterpart make PEX3 a prime drug target for the development of novel therapies against trypanosomiases. The identification paves the way for future drug development targeting PEX3, and for the analysis of additional partners involved in this crucial step of glycosome biogenesis.

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.

Life cycle of Blastocrithidia papi sp. n. (Kinetoplastea, Trypanosomatidae) in Pyrrhocoris apterus (Hemiptera, Pyrrhocoridae).

Blastocrithidia papi sp. n. is a cyst-forming trypanosomatid parasitizing firebugs (Pyrrhocoris apterus). It is a member of the Blastocrithidia clade and a very close relative of B. largi, to which it is almost identical through its SSU rRNA gene sequence. However, considering the SL RNA gene these two species represent quite distinct, not even related typing units. Morphological analysis of the new species revealed peculiar or even unique features, which may be useful for future taxonomic revision of the genus Blastocrithidia. These include a breach in the microtubular corset of rostrum at the site of contact with the flagellum, absence of desmosomes between flagellum and rostrum, large transparent vacuole near the flagellar pocket, and multiple vacuoles with fibrous content in the posterior portion of the cell. The study of the flagellates' behavior in the host intestine revealed that they may attach both to microvilli of enterocytes using swollen flagellar tip and to extracellular membranes layers using hemidesmosomes of flagellum. Laboratory experiments on B. papi transmission in P. apterus demonstrated that the parasite may be transmitted vertically (via contaminated surface of eggs) and horizontally (via contaminated substrate and/or necrophagy). We argue that the parasite exploits transmission mechanisms intended for obligate bacterial symbionts of P. apterus.

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.

Sterol Biosynthesis Pathway as an Alternative for the Anti-Protozoan Parasite Chemotherapy.

Sterols play an essential role in the physiology of eukaryotic cells; they play a pivotal role in the normal structure and function of cell membranes and also act as precursors for the synthesis of several different molecules like steroid hormones. Trypanosomatids and fungi have an essential requirement of ergosterol and other 24-alkyl sterols, which are absent in mammalian cells, for their survival and growth. At least 20 metabolic steps are necessary to synthesize sterols as cholesterol and ergosterol with the involvement of different specific enzymes. Some enzymes have been studied in detail in order to find new inhibitors that are able to abolish the parasite growth in vitro; besides, they also promote the curative efficacy in murine models of infection, thus opening new possibilities to introduce new drugs for the treatment of leishmaniasis and Chagas' disease. Sterols biosynthesis inhibitors (SBIs) can potentially be used as a chemotherapeutic agent against trypanosomatids. Actually, there are several drugs that interfere with the SB pathway, and some of them are already in clinical trials, such as posaconazole, and a new pro-drug, the ravuconazole. Furthermore, new approaches are being used, such as the combination of drugs, to reduce the resistance and minimize toxic effects. In this review, we discuss the main steps of the SB pathway, showing each enzyme involved in the steps, as well as the antiproliferative, physiological, biochemical, and ultrastructural effects of the several known inhibitors.

Molecular revision of the genus Wallaceina.

This work is focused on the molecular revision of the genus Wallaceina established in the very twilight of the classical morphotype-based approach to classification of the Trypanosomatidae. The genus was erected due to the presence of a unique variant of endomastigotes. In molecular phylogenetic studies four described species of Wallaceina were shown to be extremely close to each other and to some other undescribed isolates clustered within Leishmaniinae clade, while three recently included species formed a separate clade. Our results of morphological and molecular phylogenetic analyses demonstrated that all Leishmaniinae-bound wallaceinas are just different isolates of the same species that we rename back to Crithidia brevicula Frolov, Malysheva, 1989. To accommodate former Wallaceina spp. phylogenetically distant from the genus Crithidia, we propose a new generic name Wallacemonas Kostygov et Yurchenko, 2014.

The double-edged sword in pathogenic trypanosomatids: the pivotal role of mitochondria in oxidative stress and bioenergetics.

The pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. are the causative agents of African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. These diseases are considered to be neglected tropical illnesses that persist under conditions of poverty and are concentrated in impoverished populations in the developing world. Novel efficient and nontoxic drugs are urgently needed as substitutes for the currently limited chemotherapy. Trypanosomatids display a single mitochondrion with several peculiar features, such as the presence of different energetic and antioxidant enzymes and a specific arrangement of mitochondrial DNA (kinetoplast DNA). Due to mitochondrial differences between mammals and trypanosomatids, this organelle is an excellent candidate for drug intervention. Additionally, during trypanosomatids' life cycle, the shape and functional plasticity of their single mitochondrion undergo profound alterations, reflecting adaptation to different environments. In an uncoupling situation, the organelle produces high amounts of reactive oxygen species. However, these species role in parasite biology is still controversial, involving parasite death, cell signalling, or even proliferation. Novel perspectives on trypanosomatid-targeting chemotherapy could be developed based on better comprehension of mitochondrial oxidative regulation processes.

Structural characterization of the cell division cycle in Strigomonas culicis, an endosymbiont-bearing trypanosomatid.

Strigomonas culicis (previously referred to as Blastocrithidia culicis) is a monoxenic trypanosomatid harboring a symbiotic bacterium, which maintains an obligatory relationship with the host protozoan. Investigations of the cell cycle in symbiont harboring trypanosomatids suggest that the bacterium divides in coordination with other host cell structures, particularly the nucleus. In this study we used light and electron microscopy followed by three-dimensional reconstruction to characterize the symbiont division during the cell cycle of S. culicis. We observed that during this process, the symbiotic bacterium presents different forms and is found at different positions in relationship to the host cell structures. At the G1/S phase of the protozoan cell cycle, the endosymbiont exhibits a constricted form that appears to elongate, resulting in the bacterium division, which occurs before kinetoplast and nucleus segregation. During cytokinesis, the symbionts are positioned close to each nucleus to ensure that each daughter cell will inherit a single copy of the bacterium. These observations indicated that the association of the bacterium with the protozoan nucleus coordinates the cell cycle in both organisms.

Molecular phylogenetic redefinition of Herpetomonas (Kinetoplastea, Trypanosomatidae), a genus of insect parasites associated with flies.

In order to review the taxonomy of the genus Herpetomonas through phylogenetic and morphological analyses we barcoded 527 insect trypanosomatids by sequencing the V7V8 region of the small subunit ribosomal RNA (SSU rRNA) gene. Fifty two flagellates, 90% of them from Diptera, revealed to be related to known species of Herpetomonas. Sequences of entire glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and SSU rRNA genes were employed for phylogenetic inferences including representatives of all genera of Trypanosomatidae. In the resulting phylogenetic trees, the selected flagellates clustered into a monophyletic assemblage that we are considering as the redefined genus Herpetomonas. Internal transcribed spacer 1 (ITS1) rDNA sequences and putative secondary structures of this region were compared for evaluation of inter- and intraspecific variability. The flagellates were classified in six already known species and five new species. In addition, two Leptomonas spp. were moved to Herpetomonas, now comprising 13 valid species, while four species were excluded from the genus. Light and electron microscopy revealed the extreme polymorphism of Herpetomonas, hindering genus and species identification by morphological characteristics. Our findings also showed that some species of Herpetomonas are generalist parasites of flies and appear to be as cosmopolitan as their hosts.

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.

Effects of direct electric current on Herpetomonas samuelpessoai: An ultrastructural study.

The literature shows that the effects of direct electric currents on biological material are numerous, including bactericidal, fungicidal, parasiticidal, and anti-tumoral, among others. Non-pathogenic trypanosomatids, such as Herpetomonas samuelpessoai, have emerged as important models for the study of basic biological processes performed by a eukaryotic cell. The present study reports a dose-dependent anti-protozoan effect of direct electric treatment with both cathodic and anodic current flows on H. samuelpessoai cells. The damaging effects can be attributable to the electrolysis products generated during electric stimulation. The pH of the cell suspension was progressively augmented from 7.4 to 10.5 after the cathodic treatment. In contrast, the anodic treatment caused a pH decrease varying from 7.4 to 6.5. Transmission electron microscopy analyses revealed profound alterations in vital cellular structures (e.g., mitochondrion, kinetoplast, flagellum, flagellar pocket, nucleus, and plasma membrane) after exposure to both cathodic and anodic current flows. Specifically, cathodic current flow treatment induced the appearance of autophagic-like structures on parasite cells, while those submitted to an anodic current flow presented marked disorganization of plasma membrane and necrotic appearance. However, parasites treated in the intermediary chamber (without contact with the electrodes) did not present significant changes in viability or morphology, and no pH variation was detected in this system. The use of H. samuelpessoai as a biological model and the direct electric current experimental approach used in our study provide important information for understanding the mechanisms involved in the cytotoxic effects of this physical agent.

Morphology and growth characteristics of cultured Leptomonas ctenocephali from Ctenocephalides felis felis (Siphonaptera: Pulicidae) of dogs in Brazil.

To confirm the taxonomic identification of a trypanosomatid found in the hindgut, rectum and Malpighian tubules of dog fleas captured in Belo Horizonte, Minas Gerais, Brazil, between April and November of 2005, 910 specimens of Ctenocephalides felis felis were removed from street dogs and dissected, and isolates from their digestive tracts were cultivated in NNN-alpha-MEM medium. Four different morphological forms were observed in culture: long, slender, twisted promastigotes with a long flagellum; short, stubby, non-twisted promastigotes; rounded amastigotes; and cyst-like bodies. Twisted and non-twisted promastigotes were frequently seen forming rosettes, and these two forms presented significant differences (P<0.01) in terms of their morphological characteristics. Unlike the promastigote forms observed throughout the culture period, rounded amastigotes were seen only in the lag phase, and the cyst-like bodies were only seen in the decline phase. The trypanosomatid DNA obtained from the culture was analyzed by the polymerase chain reaction (PCR) method and found to be negative for Leishmania infantum chagasi. Based on the growth pattern, morphological parameters and molecular analysis, the flagellates were confirmed to be Leptomonas ctenocephali. The significance of this infection for animals is also commented.

Selective recovery of the cultivation-prone components from mixed trypanosomatid infections: a case of several novel species isolated from Neotropical Heteroptera.

Mixed trypanosomatid infections (a simultaneous presence of two or more parasites in the same host) have long been suspected to represent an obstacle for recovering cultures that would faithfully represent original species descriptions. However, without the means to directly compare the parasites in the host and in culture, this would remain just a possibility. Here we have used PCR-based genotyping of spliced leader RNA gene repeats to analyse several novel species of insect trypanosomatids isolated from heteropteran hosts and to compare them with the parasites that had been detected in the gut smears of the same hosts. We have found that, whereas the original infections were dominated by some blastocrithidia-like parasites, most of the respective axenic cultures contained novel species of Crithidia and Leptomonas. Therefore, we concluded that, in each case, this replacement was caused by differences in cultivation properties between the original predominant blastocrithidia and the less fastidious parasite that was later recovered in culture. The properties of the new organisms, including their morphology and ultrastructure, as well as their phylogenetic affinities within the family, were investigated and used to describe five novel species.

Evolutionary history of trypanosomes from South American caiman (Caiman yacare) and African crocodiles inferred by phylogenetic analyses using SSU rDNA and gGAPDH genes.

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDH sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.

A phylogenetic lineage of closely related trypanosomes (Trypanosomatidae, Kinetoplastida) of anurans and sand flies (Psychodidae, Diptera) sharing the same ecotopes in brazilian amazonia.

Analysis of the phylogenetic relationships among trypanosomes from vertebrates and invertebrates disclosed a new lineage of trypanosomes circulating among anurans and sand flies that share the same ecotopes in Brazilian Amazonia. This assemblage of closely related trypanosomes was determined by comparing whole SSU rDNA sequences of anuran trypanosomes from the Brazilian biomes of Amazonia, the Pantanal, and the Atlantic Forest and from Europe, North America, and Africa, and from trypanosomes of sand flies from Amazonia. Phylogenetic trees based on maximum likelihood and parsimony corroborated the positioning of all new anuran trypanosomes in the aquatic clade but did not support the monophyly of anuran trypanosomes. However, all analyses always supported four major clades (An01-04) of anuran trypanosomes. Clade An04 is composed of trypanosomes from exotic anurans. Isolates in clades An01 and An02 were from Brazilian frogs and toads captured in the three biomes studied, Amazonia, the Pantanal and the Atlantic Forest. Clade An01 contains mostly isolates from Hylidae whereas clade An02 comprises mostly isolates from Bufonidae; and clade An03 contains trypanosomes from sand flies and anurans of Bufonidae, Leptodactylidae, and Leiuperidae exclusively from Amazonia. To our knowledge, this is the first study describing morphological and growth features, and molecular phylogenetic affiliation of trypanosomes from anurans and phlebotomines, incriminating these flies as invertebrate hosts and probably also as important vectors of Amazonian terrestrial anuran trypanosomes.