Nejvíce citovaný článek - PubMed ID 22845426
New species of insect trypanosomatids from Costa Rica and the proposal for a new subfamily within the Trypanosomatidae
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.
- Klíčová slova
- Leishmania, catalase, dixeny, evolution, virulence,
- MeSH
- faktory virulence genetika metabolismus MeSH
- katalasa genetika metabolismus MeSH
- kultivované buňky MeSH
- Leishmania mexicana genetika růst a vývoj patogenita MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- protozoální proteiny genetika MeSH
- Psychodidae parazitologie MeSH
- stadia vývoje genetika MeSH
- Teschovirus genetika MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- faktory virulence MeSH
- katalasa MeSH
- protozoální proteiny MeSH
The closest relative of human pathogen Leishmania, the trypanosomatid Novymonas esmeraldas, harbors a bacterial endosymbiont "Candidatus Pandoraea novymonadis." Based on genomic data, we performed a detailed characterization of the metabolic interactions of both partners. While in many respects the metabolism of N. esmeraldas resembles that of other Leishmaniinae, the endosymbiont provides the trypanosomatid with heme, essential amino acids, purines, some coenzymes, and vitamins. In return, N. esmeraldas shares with the bacterium several nonessential amino acids and phospholipids. Moreover, it complements its carbohydrate metabolism and urea cycle with enzymes missing from the "Ca. Pandoraea novymonadis" genome. The removal of the endosymbiont from N. esmeraldas results in a significant reduction of the overall translation rate, reduced expression of genes involved in lipid metabolism and mitochondrial respiratory activity, and downregulation of several aminoacyl-tRNA synthetases, enzymes involved in the synthesis of some amino acids, as well as proteins associated with autophagy. At the same time, the genes responsible for protection against reactive oxygen species and DNA repair become significantly upregulated in the aposymbiotic strain of this trypanosomatid. By knocking out a component of its flagellum, we turned N. esmeraldas into a new model trypanosomatid that is amenable to genetic manipulation using both conventional and CRISPR-Cas9-mediated approaches. IMPORTANCENovymonas esmeraldas is a parasitic flagellate of the family Trypanosomatidae representing the closest insect-restricted relative of the human pathogen Leishmania. It bears symbiotic bacteria in its cytoplasm, the relationship with which has been established relatively recently and independently from other known endosymbioses in protists. Here, using the genome analysis and comparison of transcriptomic profiles of N. esmeraldas with and without the endosymbionts, we describe a uniquely complex cooperation between both partners on the biochemical level. We demonstrate that the removal of bacteria leads to a decelerated growth of N. esmeraldas, substantial suppression of many metabolic pathways, and increased oxidative stress. Our success with the genetic transformation of this flagellate makes it a new model trypanosomatid species that can be used for the dissection of mechanisms underlying the symbiotic relationships between protists and bacteria.
- Klíčová slova
- Leishmaniinae, Trypanosomatidae, bacterial endosymbiont, genomics, metabolism,
- MeSH
- Bacteria klasifikace genetika metabolismus MeSH
- fylogeneze MeSH
- genom bakteriální * MeSH
- genomika MeSH
- symbióza genetika MeSH
- Trypanosoma klasifikace metabolismus mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Klíčová slova
- gene gain, gene loss, genome analysis, leishmaniinae,
- MeSH
- fylogeneze MeSH
- Leishmania major klasifikace genetika MeSH
- Leishmania klasifikace genetika MeSH
- membránové proteiny genetika MeSH
- molekulární evoluce MeSH
- protozoální proteiny genetika MeSH
- regulace genové exprese MeSH
- sekvenování celého genomu metody MeSH
- stanovení celkové genové exprese MeSH
- Trypanosomatina klasifikace genetika MeSH
- virulence MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- membránové proteiny MeSH
- protozoální proteiny MeSH
Monoxenous (one host) trypanosomatids from insects and other invertebrates can be introduced into axenic culture relatively easily and efficiently, allowing for their transfer from the field into the laboratory. Here we describe simple methods and alternative cultivation protocols, the wider application of which will allow substantial expansion of trypanosomatids available for research.
- Klíčová slova
- Axenization, Cultivation, Field, Insects, Isolation, Trypanosoma, Trypanosomatids,
- MeSH
- axenická kultura metody MeSH
- hmyz parazitologie MeSH
- parazitologie přístrojové vybavení metody MeSH
- Trypanosomatina izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Trypanosomatids of the genus Leishmania are parasites of mammals or reptiles transmitted by bloodsucking dipterans. Many species of these flagellates cause important human diseases with clinical symptoms ranging from skin sores to life-threatening damage of visceral organs. The genus Leishmania contains four subgenera: Leishmania, Sauroleishmania, Viannia, and Mundinia. The last subgenus has been established recently and remains understudied, although Mundinia contains human-infecting species. In addition, it is interesting from the evolutionary viewpoint, representing the earliest branch within the genus and possibly with a different type of vector. Here we analyzed the genomes of L. (M.) martiniquensis, L. (M.) enriettii and L. (M.) macropodum to better understand the biology and evolution of these parasites. RESULTS: All three genomes analyzed were approximately of the same size (~ 30 Mb) and similar to that of L. (Sauroleishmania) tarentolae, but smaller than those of the members of subgenera Leishmania and Viannia, or the genus Endotrypanum (~ 32 Mb). This difference was explained by domination of gene losses over gains and contractions over expansions at the Mundinia node, although only a few of these genes could be identified. The analysis predicts significant changes in the Mundinia cell surface architecture, with the most important ones relating to losses of LPG-modifying side chain galactosyltransferases and arabinosyltransferases, as well as β-amastins. Among other important changes were gene family contractions for the oxygen-sensing adenylate cyclases and FYVE zinc finger-containing proteins. CONCLUSIONS: We suggest that adaptation of Mundinia to different vectors and hosts has led to alternative host-parasite relationships and, thereby, made some proteins redundant. Thus, the evolution of genomes in the genus Leishmania and, in particular, in the subgenus Mundinia was mainly shaped by host (or vector) switches.
- Klíčová slova
- L. (M.) macropodum, L. (M.) martiniquensis, Leishmania (Mundinia) enriettii, Whole genome sequencing,
- MeSH
- délka genomu MeSH
- fylogeneze MeSH
- genomika MeSH
- hostitelská specificita MeSH
- Leishmania klasifikace genetika MeSH
- molekulární evoluce MeSH
- ploidie MeSH
- protozoální proteiny genetika MeSH
- regulace genové exprese MeSH
- sekvenování celého genomu metody MeSH
- sekvenování exomu MeSH
- stanovení celkové genové exprese metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- protozoální proteiny MeSH
Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM.22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM.22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM.22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM.22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.
- MeSH
- CRISPR-Cas systémy * MeSH
- genový knockout metody MeSH
- hmyz - vektory parazitologie MeSH
- Leishmania mexicana genetika patogenita MeSH
- leishmanióza kožní parazitologie MeSH
- lidé MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- počítačová simulace MeSH
- protozoální geny * MeSH
- Psychodidae parazitologie MeSH
- stanovení celkové genové exprese MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
BACKGROUND: Leptomonas pyrrhocoris is a parasite of the firebug Pyrrhocoris apterus. This flagellate has been recently proposed as a model species for studying different aspects of the biology of monoxenous trypanosomatids, including host - parasite interactions. During its life cycle L. pyrrhocoris never tightly attaches to the epithelium of the insect gut. In contrast, its dixenous relatives (Leishmania spp.) establish a stable infection via attachment to the intestinal walls of their insect hosts. MATERIAL AND METHODS: This process is mediated by chemical modifications of the cell surface lipophosphoglycans. In our study we tested whether the inability of L. pyrrhocoris to attach to the firebug's midgut is associated with the absence of these glycoconjugates. We also analyzed evolution of the proteins involved in proper lipophosphoglycan assembly, cell attachment and establishment of a stable infection in L. pyrrhocoris, L. seymouri, and Leishmania spp. Our comparative analysis demonstrated differences in SCG/L/R repertoire between the two parasite subgenera, Leishmania and Viannia, which may be related to distinct life strategies in various Leishmania spp. The genome of L. pyrrhocoris encodes 6 SCG genes, all of which are quite divergent from their orthologs in the genus Leishmania. Using direct probing with an antibody recognizing the β-Gal side chains of lipophosphoglycans, we confirmed that these structures are not synthesized in L. pyrrhocoris. CONCLUSION: We conclude that either the SCG enzymes are not active in this species (similarly to SCG5/7 in L. major), or they possess a different biochemical activity.
- Klíčová slova
- Host-parasite interaction, Insect gut's attachment, L. pyrrhocoris, LPG, Monoxenous trypanosomatids, SCG enzymes,
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3' to 5' on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.
- MeSH
- editace RNA * MeSH
- genom mitochondriální genetika MeSH
- genom protozoální genetika MeSH
- izoformy RNA genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- RNA mitochondriální genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- sestřih RNA MeSH
- stanovení celkové genové exprese metody MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- Trypanosomatina genetika metabolismus MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- izoformy RNA MeSH
- RNA mitochondriální MeSH
- RNA protozoální MeSH
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.
- MeSH
- hmyz - vektory parazitologie MeSH
- Leishmania mexicana genetika patogenita MeSH
- leishmanióza kožní parazitologie MeSH
- makrofágy parazitologie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- proteiny vázající GTP genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- Psychodidae parazitologie MeSH
- virulence MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- proteiny vázající GTP MeSH
- protozoální proteiny MeSH
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.
