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

Ishemgulova, Aygul; Kraeva, Natalya; Faktorová, Drahomíra; Podešvová, Lucie; Lukeš, Julius; Yurchenko, Vyacheslav

doi:10.14411/fp.2016.016

Cited by 13 publications

(17 citation statements)

References 29 publications

(29 reference statements)

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“…36 . 1140 (gene encoding a short chain 3-hydroxyacyl-CoA dehydrogenase) were used [ 28 ]. Quantitative PCR analysis (RT-qPCR) was performed as described previously [ 29 ].…”

Section: Methodsmentioning

confidence: 99%

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CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1

et al. 2018

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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.

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“…36 . 1140 (gene encoding a short chain 3-hydroxyacyl-CoA dehydrogenase) were used [ 28 ]. Quantitative PCR analysis (RT-qPCR) was performed as described previously [ 29 ].…”

Section: Methodsmentioning

confidence: 99%

“…L . mexicana expressing mCherry [ 28 ] was transfected with 5 μg of linearized resulting plasmid and clones of L . mexicana -mCherry-Cas9 were selected on solid M199 medium supplemented as above with additional 100 μg/ml of Hyg.…”

Section: Methodsmentioning

confidence: 99%

CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1

et al. 2018

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“…36 . 1140 (gene encoding a short chain 3-hydroxyacyl-CoA dehydrogenase) were used [24]. Quantitative PCR analysis (RT-qPCR) was performed as described previously [25].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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BackgroundLeishmania 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 findingsThe 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/SignificanceL. 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.

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“…Mouse bone-marrow derived macrophages were infected as described previously [63] with modifications [64]. In brief, differentiated macrophages were cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 50 units/mL of penicillin, 50 µg/mL of streptomycin, 2 mM of L-glutamine, and 0.05 mM of 2-mercapto-ethanol (all from Sigma-Aldrich) at 37 • C with 5% CO 2 .…”

Section: Macrophage Infectionmentioning

confidence: 99%

The First Non-LRV RNA Virus in Leishmania

Grybchuk

Macedo

Kleschenko

et al. 2020

Viruses

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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 Leishmania martiniquensis, 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.

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

Cited by 13 publications

References 29 publications

CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1

CRISPR/Cas9 in Leishmania mexicana: A case study of LmxBTN1

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

The First Non-LRV RNA Virus in Leishmania

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