The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.
- MeSH
- buněčné dělení MeSH
- buněčný cyklus MeSH
- Leishmania mexicana * genetika MeSH
- Leishmania * MeSH
- paraziti * MeSH
- Psychodidae * parazitologie MeSH
- savci MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. CONCLUSION/SIGNIFICANCE: Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.
- MeSH
- antigen Ku genetika metabolismus MeSH
- genom protozoální MeSH
- Leishmania mexicana genetika metabolismus MeSH
- leishmanióza kožní parazitologie MeSH
- lidé MeSH
- nestabilita genomu * MeSH
- protozoální proteiny genetika metabolismus MeSH
- telomery genetika metabolismus MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Telomeres are the ends of linear eukaryotic chromosomes facilitating the resolution of the ‘end replication and protection' problems, associated with linearity. At the nucleotide level, telomeres typically represent stretches of tandemly arranged telomeric repeats, which vary in length and sequence among different groups of organisms. Recently, a composition of the telomere-associated protein complex has been scrutinized in Trypanosoma brucei. In this work, we subjected proteins from that list to a more detailed bioinformatic analysis and delineated a core set of 20 conserved proteins putatively associated with telomeres in trypanosomatids. Out of these, two proteins (Ku70 and Ku80) are conspicuously missing in representatives of the genus Blastocrithidia, yet telomeres in these species do not appear to be affected. In this work, based on the analysis of a large set of trypanosomatids widely different in their phylogenetic position and life strategies, we demonstrated that telomeres of trypanosomatids are diverse in length, even within groups of closely related species. Our analysis showed that the expression of two proteins predicted to be associated with telomeres (those encoding telomerase and telomere-associated hypothetical protein orthologous to Tb927.6.4330) may directly affect and account for the differences in telomere length within the species of the Leishmania mexicana complex.
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.
- 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
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: 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
Targeted regulation of protein levels is an important tool to investigate the role of proteins essential for cell function and development. In recent years, methods based on the Escherichia coli dihydrofolate reductase destabilization domain (ecDHFR DD) have been established and used in various cell types. ecDHFR DD destabilizes the fused protein of interest and causes its degradation by proteasomes, unless it is stabilized by a specific ligand, trimethoprim. In this work we developed an inducible protein stabilization system in Leishmania mexicana based on ecDHFR DD.
- MeSH
- aktivace transkripce * MeSH
- dihydrofolátreduktasa genetika metabolismus MeSH
- Escherichia coli enzymologie genetika MeSH
- Leishmania mexicana genetika metabolismus MeSH
- molekulární biologie metody MeSH
- parazitologie metody MeSH
- regulace genové exprese * MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- trimethoprim metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
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.
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.
