Complete minicircle genome of Leptomonas pyrrhocoris reveals sources of its non-canonical mitochondrial RNA editing events.

Uridine insertion/deletion (U-indel) editing of mitochondrial mRNA, unique to the protistan class Kinetoplastea, generates canonical as well as potentially non-productive editing events. While the molecular machinery and the role of the guide (g) RNAs that provide required information for U-indel editing are well understood, little is known about the forces underlying its apparently error-prone nature. Analysis of a gRNA:mRNA pair allows the dissection of editing events in a given position of a given mitochondrial transcript. A complete gRNA dataset, paired with a fully characterized mRNA population that includes non-canonically edited transcripts, would allow such an analysis to be performed globally across the mitochondrial transcriptome. To achieve this, we have assembled 67 minicircles of the insect parasite Leptomonas pyrrhocoris, with each minicircle typically encoding one gRNA located in one of two similar-sized units of different origin. From this relatively narrow set of annotated gRNAs, we have dissected all identified mitochondrial editing events in L. pyrrhocoris, the strains of which dramatically differ in the abundance of individual minicircle classes. Our results support a model in which a multitude of editing events are driven by a limited set of gRNAs, with individual gRNAs possessing an inherent ability to guide canonical and non-canonical editing.

Leptomonas seymouri: Adaptations to the Dixenous Life Cycle Analyzed by Genome Sequencing, Transcriptome Profiling and Co-infection with Leishmania donovani.

The co-infection cases involving dixenous Leishmania spp. (mostly of the L. donovani complex) and presumably monoxenous trypanosomatids in immunocompromised mammalian hosts including humans are well documented. The main opportunistic parasite has been identified as Leptomonas seymouri of the sub-family Leishmaniinae. The molecular mechanisms allowing a parasite of insects to withstand elevated temperature and substantially different conditions of vertebrate tissues are not understood. Here we demonstrate that L. seymouri is well adapted for the environment of the warm-blooded host. We sequenced the genome and compared the whole transcriptome profiles of this species cultivated at low and high temperatures (mimicking the vector and the vertebrate host, respectively) and identified genes and pathways differentially expressed under these experimental conditions. Moreover, Leptomonas seymouri was found to persist for several days in two species of Phlebotomus spp. implicated in Leishmania donovani transmission. Despite of all these adaptations, L. seymouri remains a predominantly monoxenous species not capable of infecting vertebrate cells under normal conditions.

T7 polymerase-driven transcription is downregulated in metacyclic promastigotes and amastigotes of Leishmania mexicana.

In our previous work we established a T7 polymerase-driven Tetracycline-inducible protein expression system in Leishmania mexicana (Biagi, 1953). We used this system to analyse gene expression profiles during development of L. mexicana in procyclic and metacyclic promastigotes and amastigotes. The transcription of the gene of interest and the T7 polymerase genes was significantly reduced upon cell differentiation. This regulation is not locus-specific. It depends on untranslated regions flanking open reading frames of the genes analysed. In this paper, we report that the previously established conventional inducible protein expression system may not be suitable for studies on differentiation of species of Leishmania Ross, 1903 and protein expression systems might have certain limitations.

Ultrastructure and molecular phylogeny of four new species of monoxenous trypanosomatids from flies (Diptera: Brachycera) with redefinition of the genus Wallaceina.

Four new species of monoxenous kinetoplastid parasites are described from Brachycera flies, namely Wallaceina raviniae Votýpka et Lukes, 2014 and Crithidia otongatchiensis Votýpka et Lukes, 2014 from Ecuador, Leptomonas moramango Votypka et Lukes, 2014 from Madagascar, and Crithidia pragensis Votýpka, Klepetková et Lukes, 2014 from the Czech Republic. The new species are described here based on sequence analysis of their spliced leader (SL) RNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and small subunit (SSU) rRNA genes, as well as their morphology and ultrastructure. High-pressure freezing and Bernhard's EDTA regressive staining, used for the first time for monoxenous (one host) trypanosomatids, revealed the presence of viral particles with cytosolic localization in one and unique mitochondrial localization in another species. In accordance with previous observations, our results emphasize a discrepancy between morphology and molecular taxonomy of the family Trypanosomatidae. All four newly described species are represented by typical morphotypes (mainly choano- and promastigotes) and are virtually indistinguishable from other monoxenous trypanosomatids by morphology. Nevertheless, they all differ in their phylogenetic affinities. Whereas three of them grouped within the recently defined subfamily Leishmaniinae, which includes numerous representatives of the genera Leishmania Ross, 1903, Crithidia Léger, 1902 and Leptomonas Kent, 1880, the fourth species clusters together with the 'collosoma' clade (named after 'Leptomonas' collosoma Wallace, Clark, Dyer et Collins, 1960). Here we demonstrate that the 'collosoma' group represents the elusive genus Wallaceina Podlipaev, Frolov et Kolesnikov, 1999. We redefine this genus in molecular terms based on similarities of the respective molecular markers and propose to use this taxon name for the group of species of the 'collosoma' clade.

Intricate balance of dually-localized catalase modulates infectivity of Leptomonas seymouri (Kinetoplastea: Trypanosomatidae).

Nearly all aerobic organisms are equipped with catalases, powerful enzymes scavenging hydrogen peroxide and facilitating defense against harmful reactive oxygen species. In trypanosomatids, this enzyme was not present in the common ancestor, yet it had been independently acquired by different lineages of monoxenous trypanosomatids from different bacteria at least three times. This observation posited an obvious question: why was catalase so "sought after" if many trypanosomatid groups do just fine without it? In this work, we analyzed subcellular localization and function of catalase in Leptomonas seymouri. We demonstrated that this enzyme is present in the cytoplasm and a subset of glycosomes, and that its cytoplasmic retention is H2O2-dependent. The ablation of catalase in this parasite is not detrimental in vivo, while its overexpression resulted in a substantially higher parasite load in the experimental infection of Dysdercus peruvianus. We propose that the capacity of studied flagellates to modulate the catalase activity in the midgut of its insect host facilitates their development and protects them from oxidative damage at elevated temperatures.

Comparative Analysis of Three Trypanosomatid Catalases of Different Origin.

Most trypanosomatid flagellates do not have catalase. In the evolution of this group, the gene encoding catalase has been independently acquired at least three times from three different bacterial groups. Here, we demonstrate that the catalase of Vickermania was obtained by horizontal gene transfer from Gammaproteobacteria, extending the list of known bacterial sources of this gene. Comparative biochemical analyses revealed that the enzymes of V. ingenoplastis, Leptomonas pyrrhocoris, and Blastocrithidia sp., representing the three independent catalase-bearing trypanosomatid lineages, have similar properties, except for the unique cyanide resistance in the catalase of the latter species.

Catalase impairs Leishmania mexicana development and virulence.

Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the Leishmania mexicana Β-TUBULIN locus using a novel bicistronic expression system, which relies on the 2A peptide of Teschovirus A. We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of Leishmania, resulting in loss of this gene from the genome during the evolution of these parasites.

Common Structural Patterns in the Maxicircle Divergent Region of Trypanosomatidae.

Maxicircles of all kinetoplastid flagellates are functional analogs of mitochondrial genome of other eukaryotes. They consist of two distinct parts, called the coding region and the divergent region (DR). The DR is composed of highly repetitive sequences and, as such, remains the least explored segment of a trypanosomatid genome. It is extremely difficult to sequence and assemble, that is why very few full length maxicircle sequences were available until now. Using PacBio data, we assembled 17 complete maxicircles from different species of trypanosomatids. Here we present their large-scale comparative analysis and describe common patterns of DR organization in trypanosomatids.

The First Non-LRV RNA Virus in Leishmania.

In this work, we describe the first Leishmania-infecting leishbunyavirus-the first virus other than Leishmania RNA virus (LRV) found in trypanosomatid parasites. Its host is Leishmaniamartiniquensis, a human pathogen causing infections with a wide range of manifestations from asymptomatic to severe visceral disease. This virus (LmarLBV1) possesses many characteristic features of leishbunyaviruses, such as tripartite organization of its RNA genome, with ORFs encoding RNA-dependent RNA polymerase, surface glycoprotein, and nucleoprotein on L, M, and S segments, respectively. Our phylogenetic analyses suggest that LmarLBV1 originated from leishbunyaviruses of monoxenous trypanosomatids and, probably, is a result of genomic re-assortment. The LmarLBV1 facilitates parasites' infectivity in vitro in primary murine macrophages model. The discovery of a virus in L.martiniquensis poses the question of whether it influences pathogenicity of this parasite in vivo, similarly to the LRV in other Leishmania species.

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.

LmxM.22.0250-Encoded Dual Specificity Protein/Lipid Phosphatase Impairs Leishmania mexicana Virulence In Vitro.

Protein phosphorylation/dephosphorylation is an important regulatory mechanism that controls many key physiological processes. Numerous pathogens successfully use kinases and phosphatases to internalize, replicate, and survive, modifying the host's phosphorylation profile or signal transduction pathways. Multiple phosphatases and kinases from diverse bacterial pathogens have been implicated in human infections before. In this work, we have identified and characterized the dual specificity protein/lipid phosphatase LmDUSP1 as a novel virulence factor governing Leishmania mexicana infection. The LmDUSP1-encoding gene (LmxM.22.0250 in L. mexicana) has been acquired from bacteria via horizontal gene transfer. Importantly, its orthologues have been associated with virulence in several bacterial species, such as Mycobacterium tuberculosis and Listeria monocytogenes. Leishmania mexicana with ablated LmxM.22.0250 demonstrated severely attenuated virulence in the experimental infection of primary mouse macrophages, suggesting that this gene facilitates Leishmania pathogenicity in vertebrates. Despite significant upregulation of LmxM.22.0250 expression in metacyclic promastigotes, its ablation did not affect the ability of mutant cells to differentiate into virulent stages in insects. It remains to be further investigated which specific biochemical pathways involve LmDUSP1 and how this facilitates the parasite's survival in the host. One of the interesting possibilities is that LmDUSP1 may target host's substrate(s), thereby affecting its signal transduction pathways.

Catalase in Leishmaniinae: With me or against me?

The catalase gene is a virtually ubiquitous component of the eukaryotic genomes. It is also present in the monoxenous (i.e. parasitizing solely insects) trypanosomatids of the subfamily Leishmaniinae, which have acquired the enzyme by horizontal gene transfer from a bacterium. However, as shown here, the catalase gene was secondarily lost from the genomes of all Leishmania sequenced so far. Due to the potentially key regulatory role of hydrogen peroxide in the inter-stagial transformation of Leishmania spp., this loss seems to be a necessary prerequisite for the emergence of a complex life cycle of these important human pathogens. Hence, in this group of protists, the advantages of keeping catalase were uniquely outweighed by its disadvantages.

A putative ATP/GTP binding protein affects Leishmania mexicana growth in insect vectors and vertebrate hosts.

BACKGROUND: Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania's ability to establish mammalian intracellular infection and to colonize the gut of an insect vector. METHODOLOGY/PRINCIPAL FINDINGS: The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed. CONCLUSIONS/SIGNIFICANCE: L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana's general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.

Molecular mechanisms of thermal resistance of the insect trypanosomatid Crithidia thermophila.

In the present work, we investigated molecular mechanisms governing thermal resistance of a monoxenous trypanosomatid Crithidia luciliae thermophila, which we reclassified as a separate species C. thermophila. We analyzed morphology, growth kinetics, and transcriptomic profiles of flagellates cultivated at low (23°C) and elevated (34°C) temperature. When maintained at high temperature, they grew significantly faster, became shorter, with genes involved in sugar metabolism and mitochondrial stress protection significantly upregulated. Comparison with another thermoresistant monoxenous trypanosomatid, Leptomonas seymouri, revealed dramatic differences in transcription profiles of the two species with only few genes showing the same expression pattern. This disparity illustrates differences in the biology of these two parasites and distinct mechanisms of their thermotolerance, a prerequisite for living in warm-blooded vertebrates.

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.

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.

Tetracycline-inducible gene expression system in Leishmania mexicana.

Here we present a T7-driven, tetracycline-inducible system for protein expression in human pathogen Leishmania mexicana. The gene expression in this strain is tightly regulated and dose- and time-dependent. This system can be widely used by the parasitology community to analyze effects of genes of interest on biology, physiology and virulence of parasites causing cutaneous leishmaniases.

Kentomonas gen. n., a new genus of endosymbiont-containing trypanosomatids of Strigomonadinae subfam. n.

Compared to their relatives, the diversity of endosymbiont-containing Trypanosomatidae remains under-investigated, with only two new species described in the past 25 years, bringing the total to six. The possible reasons for such a poor representation of this group are either their overall scarcity or susceptibility of their symbionts to antibiotics that are traditionally used for cultivation of flagellates. In this work we describe the isolation, cultivation, as well as morphological and molecular characterization of a novel endosymbiont-harboring trypanosomatid species, Kentomonas sorsogonicus sp. n. The newly erected genus Kentomonas gen. n. shares many common features with the genera Angomonas and Strigomonas, such as the presence of an extensive system of peripheral mitochondrial branches distorting the corset of subpellicular microtubules, large and loosely packed kinetoplast, and a rudimentary paraflagellar rod. Here we also propose to unite all endosymbiont-bearing trypanosomatids into the new subfamily Strigomonadinae subfam. n.

Diversity of trypanosomatids (Kinetoplastea: Trypanosomatidae) parasitizing fleas (Insecta: Siphonaptera) and description of a new genus Blechomonas gen. n.

To further investigate the diversity of Trypanosomatidae we have examined the species present within the flea (Siphonaptera) population in the Czech Republic. Out of 1549 fleas, 239 were found to be infected by trypanosomatids. Axenic cultures were established from 90 infected specimens and 29 of them were further characterized. Molecular phylogenetic analysis of the SL RNA, gGAPDH, and SSU rRNA genes revealed a striking diversity within this group and analyzed isolates were classified into 16 Typing units (TUs) of which 15 typified new species. In addition to one Trypanosoma species, two TUs grouped within the sub-family Leishmaniinae, two clustered together with Herpetomonas, wheras 11 TUs formed a novel clade branching off between Trypanosoma spp. and remaining trypanosomatids. We propose to recognize this clade as a new genus Blechomonas and a new subfamily Blechomonadinae, and provide molecular and morphological description of 11 TUs representing this genus. Our finding of such an ancient host-specific group sheds new light at the origin of Trypanosomatidae and the roots of dixenous parasitism. The strict host restriction of Blechomonas to Siphonaptera with adult fleas' dependence on blood meal may reflect passing of parasites from larvae through pupae to adults and implies potential transmission to the warm-blooded vertebrates.