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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]

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. [ABSTRACT FROM AUTHOR]

Protozoan pathogens that cause leishmaniasis in humans are relatively refractory to genetic manipulation. In this work, we implemented the CRISPR- Cas9 system in L eishmania 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. [ABSTRACT FROM AUTHOR]

The mechanics of DNA replication and cell cycling are well-characterized in model organisms, but less is known about these basic aspects of cell biology in early-diverging Apicomplexan parasites, which do not divide by canonical binary fission but undergo unconventional cycles. Schizogony in the malaria parasite, Plasmodium, generates ~16-24 new nuclei via independent, asynchronous rounds of genome replication prior to cytokinesis and little is known about the control of DNA replication that facilitates this. We have characterised replication dynamics in P. falciparum throughout schizogony, using DNA fibre labelling and combing to visualise replication forks at a single-molecule level. We show that origins are very closely spaced in Plasmodium compared to most model systems, and that replication dynamics vary across the course of schizogony, from faster synthesis rates and more widely-spaced origins through to slower synthesis rates and closer-spaced origins. This is the opposite of the pattern usually seen across S-phase in human cells, when a single genome is replicated. Replication forks also appear to stall at an unusually high rate throughout schizogony. Our work explores Plasmodium DNA replication in unprecedented detail and opens up tremendous scope for analysing cell cycle dynamics and developing interventions targetting this unique aspect of malaria biology.

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.)