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Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania., (© 2020 John Wiley & Sons Ltd.)

Leishmania and Trypanosoma are unicellular parasites that possess markedly original biological features as compared to other eukaryotes. The Leishmania genome displays a constitutive 'mosaic aneuploidy', whereas in Trypanosoma brucei, the megabase-sized chromosomes are diploid. We accurately analysed DNA replication parameters in three Leishmania species and Trypanosoma brucei as well as mouse embryonic fibroblasts (MEF). Active replication origins were visualized at the single molecule level using DNA molecular combing. More than one active origin was found on most DNA fibres, showing that the chromosomes are replicated from multiple origins. Inter-origin distances (IODs) were measured and found very large in trypanosomatids: the mean IOD was 160 kb in T. brucei and 226 kb in L. mexicana. Moreover, the progression of replication forks was faster than in any other eukaryote analyzed so far (mean velocity 1.9 kb/min in T. brucei and 2.4-2.6 kb/min in Leishmania). The estimated total number of active DNA replication origins in trypanosomatids is ~170. Finally, 14.4% of unidirectional replication forks were observed in T. brucei, in contrast to 1.5-1.7% in Leishmania and 4% in MEF cells. The biological significance of these original features is discussed.

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR-Cas9 system in Leishmania parasites and demonstrated its efficient use for genome editing. The Cas9 endonuclease was expressed under the control of the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) promoter and the single guide RNA was produced under the control of the U6snRNA promoter and terminator. As a proof of concept, we chose to knockout a tandemly repeated gene family, the paraflagellar rod-2 locus. We were able to obtain null mutants in a single round of transfection. In addition, we confirmed the absence of off-target editions by whole genome sequencing of two independent clones. Our work demonstrates that CRISPR-Cas9-mediated gene knockout represents a major improvement in comparison with existing methods. Beyond gene knockout, this genome editing tool opens avenues for a multitude of functional studies to speed up research on leishmaniasis., (© 2015 John Wiley & Sons Ltd.)

Using fluorescence in situ hybridization, we determined the ploidy of four species of Leishmania: Leishmania infantum, Leishmania donovani, Leishmania tropica and Leishmania amazonensis. We found that each cell in a strain possesses a combination of mono-, di- and trisomies for all chromosomes; ploidy patterns were different among all strains/species. These results extend those we previously described in Leishmania major, demonstrating that mosaic aneuploidy is a genetic feature widespread to the Leishmania genus. In addition to the genetic consequences induced by this mosaicism, the apparent absence of alternation between haploid/diploid stages questions the modality of genetic exchange in Leishmania sp., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania. Author summary: Leishmania are flagellated trypanosomatid parasites causing worldwide human and animal diseases. As 'divergent eukaryotes', their biology presents unique features and structures, of which the specific functions constitute potential drug targets. Among others, they possess a unique cytoskeletal structure termed the flagellum attachment zone (FAZ) attaching the base of their flagellum to one side of the flagellar pocket (FP), which is the sole site for endocytosis and exocytosis. The FP together with other unique flagellum-associated structures are crucial for parasite survival, but the functioning of this whole remains largely enigmatic. Leishmania also possess an expanded repertoire of kinesins (>55), including two trypanosomatid-specific families. Here, we show that the deletion of the sole kinesin among FAZ proteins disrupts cell morphogenesis, FP organisation and cell division. Furthermore, the ability to proliferate in the insect vector and mammalian host is reduced in parasites lacking the kinesin FAZ7B. This study helps elucidate the factors contributing to the successful lifecycle and pathogenicity of the parasite. It also highlights the functional diversification of motor proteins during evolution. [ABSTRACT FROM AUTHOR]