The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis, cell division and virulence in the mammalian host.

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.

Universal highly efficient conditional knockout system in Leishmania, with a focus on untranscribed region preservation.

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.

Identification of the centromeres of Leishmania major: revealing the hidden pieces.

Leishmania affects millions of people worldwide. Its genome undergoes constitutive mosaic aneuploidy, a type of genomic plasticity that may serve as an adaptive strategy to survive distinct host environments. We previously found high rates of asymmetric chromosome allotments during mitosis that lead to the generation of such ploidy. However, the underlying molecular events remain elusive. Centromeres and kinetochores most likely play a key role in this process, yet their identification has failed using classical methods. Our analysis of the unconventional kinetochore complex recently discovered in Trypanosoma brucei (KKTs) leads to the identification of a Leishmania KKT gene candidate (LmKKT1). The GFP-tagged LmKKT1 displays "kinetochore-like" dynamics of intranuclear localization throughout the cell cycle. By ChIP-Seq assay, one major peak per chromosome is revealed, covering a region of 4 ±2 kb. We find two largely conserved motifs mapping to 14 of 36 chromosomes while a higher density of retroposons are observed in 27 of 36 centromeres. The identification of centromeres and of a kinetochore component of Leishmania chromosomes opens avenues to explore their role in mosaic aneuploidy.

The nucleoporin Mlp2 is involved in chromosomal distribution during mitosis in trypanosomatids.

Nucleoporins are evolutionary conserved proteins mainly involved in the constitution of the nuclear pores and trafficking between the nucleus and cytoplasm, but are also increasingly viewed as main actors in chromatin dynamics and intra-nuclear mitotic events. Here, we determined the cellular localization of the nucleoporin Mlp2 in the 'divergent' eukaryotes Leishmania major and Trypanosoma brucei. In both protozoa, Mlp2 displayed an atypical localization for a nucleoporin, essentially intranuclear, and preferentially in the periphery of the nucleolus during interphase; moreover, it relocated at the mitotic spindle poles during mitosis. In T. brucei, where most centromeres have been identified, TbMlp2 was found adjacent to the centromeric sequences, as well as to a recently described unconventional kinetochore protein, in the periphery of the nucleolus, during interphase and from the end of anaphase onwards. TbMlp2 and the centromeres/kinetochores exhibited a differential migration towards the poles during mitosis. RNAi knockdown of TbMlp2 disrupted the mitotic distribution of chromosomes, leading to a surprisingly well-tolerated aneuploidy. In addition, diploidy was restored in a complementation assay where LmMlp2, the orthologue of TbMlp2 in Leishmania, was expressed in TbMlp2-RNAi-knockdown parasites. Taken together, our results demonstrate that Mlp2 is involved in the distribution of chromosomes during mitosis in trypanosomatids.

First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites.

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.

TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals.

TbFlabarin is the Trypanosoma brucei orthologue of the Leishmania flagellar protein LdFlabarin but its sequence is 33% shorter than LdFlabarin, as it lacks a C-terminal domain that is indispensable for LdFlabarin to localize to the Leishmania flagellum. TbFlabarin is mainly expressed in the procyclic forms of the parasite and localized to the flagellum, but only when two palmitoylable cysteines at positions 3 and 4 are present. TbFlabarin is more strongly attached to the membrane fraction than its Leishmania counterpart, as it resists complete solubilization with as much as 0.5% NP-40. Expression ablation by RNA interference did not change parasite growth in culture, its morphology or apparent motility. Heterologous expression showed that neither TbFlabarin in L. amazonensis nor LdFlabarin in T. brucei localized to the flagellum, revealing non-cross-reacting targeting signals between the two species.

RNA interference screen reveals a high proportion of mitochondrial proteins essential for correct cell cycle progress in Trypanosoma brucei.

BACKGROUND: Trypanosomatid parasites possess a single mitochondrion which is classically involved in the energetic metabolism of the cell, but also, in a much more original way, through its single and complex DNA (termed kinetoplast), in the correct progress of cell division. In order to identify proteins potentially involved in the cell cycle, we performed RNAi knockdowns of 101 genes encoding mitochondrial proteins using procyclic cells of Trypanosoma brucei. RESULTS: A major cell growth reduction was observed in 10 cases and a moderate reduction in 29 other cases. These data are overall in agreement with those previously obtained by a case-by-case approach performed on chromosome 1 genes, and quantitatively with those obtained by "high-throughput phenotyping using parallel sequencing of RNA interference targets" (RIT-seq). Nevertheless, a detailed analysis revealed many qualitative discrepancies with the RIT-seq-based approach. Moreover, for 37 out of 39 mutants for which a moderate or severe growth defect was observed here, we noted abnormalities in the cell cycle progress, leading to increased proportions of abnormal cell cycle stages, such as cells containing more than 2 kinetoplasts (K) and/or more than 2 nuclei (N), and modified proportions of the normal phenotypes (1N1K, 1N2K and 2N2K). CONCLUSIONS: These data, together with the observation of other abnormal phenotypes, show that all the corresponding mitochondrial proteins are involved, directly or indirectly, in the correct progress or, less likely, in the regulation, of the cell cycle in T. brucei. They also show how post-genomics analyses performed on a case-by-case basis may yield discrepancies with global approaches.

Novel insights into genome plasticity in Eukaryotes: mosaic aneuploidy in Leishmania.

Leishmania are unicellular eukaryotes that have many markedly original molecular features compared with other uni- or multicellular eukaryotes like yeasts or mammals. Genome plasticity in this parasite has been the subject of many publications, and has been associated with drug resistance or adaptability. Aneuploidy has been suspected by several authors and it is now confirmed using state-of-the-art technologies such as high-throughput DNA sequencing. The analysis of genome contents at the single cell level using fluorescence in situ hybridization (FISH) has brought a new light on the genome organization: within a cell population, every chromosome, in every cell, may be present in at least two ploidy states (being either monosomic, disomic or trisomic), and the chromosomal content varies greatly from cell to cell, thus generating a constitutive intra-strain genomic heterogeneity, here termed 'mosaic aneuploidy'. Mosaic aneuploidy deeply affects the genetics of these organisms, leading, for example, to an extreme degree of intra-strain genomic diversity, as well as to a clearance of heterozygous cells in the population without however affecting genetic heterogeneity. Second, mosaic aneuploidy might be considered as a powerful strategy evolved by the parasite for adapting to modifications of environment conditions as well as for the emergence of drug resistance. On the whole, mosaic aneuploidy may be considered as a novel mechanism for generating phenotypic diversity driven by genomic plasticity.

FISH analysis reveals aneuploidy and continual generation of chromosomal mosaicism in Leishmania major.

The protozoan parasite Leishmania is generally considered to be diploid, although a few chromosomes have been described as aneuploid. Using fluorescence in situ hybridization (FISH), we determined the number of homologous chromosomes per individual cell in L. major (i) during interphase and (ii) during mitosis. We show that, in Leishmania, aneuploidy appears to be the rule, as it affects all the chromosomes that we studied. Moreover, every chromosome was observed in at least two ploidy states, among monosomic, disomic or trisomic, in the cell population. This variable chromosomal ploidy among individual cells generates intra-strain heterogeneity, here precisely chromosomal mosaicism. We also show that this mosaicism, hence chromosome ploidy distribution, is variable among clones and strains. Finally, when we examined dividing nuclei, we found a surprisingly high rate of asymmetric chromosome allotments, showing that the transmission of genetic material during mitosis is highly unstable in this 'divergent' eukaryote: this leads to continual generation of chromosomal mosaicism. Using these results, we propose a model for the occurrence and persistence of this mosaicism. We discuss the implications of this additional unique feature of Leishmania for its biology and genetics, in particular as a novel genetic mechanism to generate phenotypic variability from genomic plasticity.

A novel microtubule-depolymerizing kinesin involved in length control of a eukaryotic flagellum.

Cilia and flagella are complex, microtubule (MT)-filled cell organelles of which the structure is evolutionarily conserved from protistan cells to mammalian sperm and the size is regulated. The best-established model for flagellar length (FL) control is set by the balance of continuous MT assembly and disassembly occurring at the flagellar tip. Because steady-state assembly of tubulin onto the distal end of the flagellum requires intraflagellar transport (IFT)--a bidirectional movement of large protein complexes that occurs within the flagellum--FL control must rely upon the regulation of IFT. This does not preclude that other pathways might "directly" affect MT assembly and disassembly. Now, among the superfamily of kinesins, family-13 (MCAK/KIF2) members exhibit a MT-depolymerizing activity responsible for their essential functions in mitosis. Here we present a novel family-13 kinesin from the flagellated protozoan parasite Leishmania major, that localizes essentially to the flagellum, and whose overexpression produces flagellar shortening and knockdown yields long flagella. Using negative mutants, we demonstrate that this phenotype is linked with the MT-binding and -depolymerizing activity of this kinesin. This is the first report of an effector protein involved in FL control through a direct action in MT dynamics, thus this finding complements the assembly-disassembly model.

Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids.

Microtubules are key players in the biology of Trypanosomatid parasites, not only as classical components of the mitotic spindle, microtubule-organizing centres and flagellum but also as the essential constituent of the cytoskeleton. Their length dynamics are regulated by, among others, microtubule-severing proteins. Four and six genes encoding microtubule-severing proteins can be found bioinformatically in the Leishmania major and Trypanosoma brucei genome respectively. We investigated all these proteins in these organisms, which include the katanin, katanin-like, spastin and fidgetin, and looked at their subcellular localization as well as their putative function by examining 'loss-of-function' phenotypes. The katanin-like KAT60b was found implicated in flagellar length reduction, but not in its size increase, while the katanin p80 subunit appeared clearly involved in cytokinesis. Fidgetin and spastin homologues were both localized in the nucleus: the first as a discrete and variable number of dots during most of the cell cycle, redistributing to the spindle and midbody during mitosis; the second concentrated as < or = 5 perinucleolar punctuations, similar to the electron-dense plaques identified in T. brucei, which were assimilated to kinetochores. This first study of microtubule-severing proteins in 'divergent' eukaryotes gives further insight into the multiple functions of these proteins identified in the hitherto studied models.

Compared genomics of the strand switch region of Leishmania chromosome 1 reveal a novel genus-specific gene and conserved structural features and sequence motifs.

BACKGROUND: Trypanosomatids exhibit a unique gene organization into large directional gene clusters (DGCs) in opposite directions. The transcription "strand switch region" (SSR) separating the two large DGCs that constitute chromosome 1 of Leishmania major has been the subject of several studies and speculations. Thus, it has been suspected of being the single replication origin of the chromosome, the transcription initiation site for both DGCs or even a centromere. Here, we have used an inter-species compared genomics approach on this locus in order to try to identify conserved features or motifs indicative of a putative function. RESULTS: We isolated, and compared the structure and nucleotide sequence of, this SSR in 15 widely divergent species of Leishmania and Sauroleishmania. As regards its intrachromosomal position, size and AT content, the general structure of this SSR appears extremely stable among species, which is another demonstration of the remarkable structural stability of these genomes at the evolutionary level. Sequence alignments showed several interesting features. Overall, only 30% of nucleotide positions were conserved in the SSR among the 15 species, versus 74% and 62% in the 5' parts of the adjacent XPP and PAXP genes, respectively. However, nucleotide divergences were not distributed homogeneously along this sequence. Thus, a central fragment of approximately 440 bp exhibited 54% of identity among the 15 species. This fragment actually represents a new Leishmania-specific CDS of unknown function which had been overlooked since the annotation of this chromosome. The encoded protein comprises two trans-membrane domains and is classified in the "structural protein" GO category. We cloned this novel gene and expressed it as a recombinant green fluorescent protein-fused version, which showed its localisation to the endoplasmic reticulum. The whole of these data shorten the actual SSR to an 887-bp segment as compared with the original 1.6 kb. In the rest of the SSR, the percentage of identity was much lower, around 22%. Interestingly, the 72-bp fragment where the putatively single transcription initiation site of chromosome 1 was identified is located in a low-conservation portion of the SSR and is itself highly polymorphic amongst species. Nevertheless, it is highly C-rich and presents a unique poly(C) tract in the same position in all species. CONCLUSION: This inter-specific comparative study, the first of its kind, (a) allowed to reveal a novel genus-specific gene and (b) identified a conserved poly(C) tract in the otherwise highly polymorphic region containing the putative transcription initiation site. This allows hypothesising an intervention of poly(C)-binding proteins known elsewhere to be involved in transcriptional control.

The switch region on Leishmania major chromosome 1 is not required for mitotic stability or gene expression, but appears to be essential.

The Leishmania genome project reference strain, Leishmania major Friedlin, is trisomic for chromosome 1. The complete sequence of this chromosome has revealed that the genes are grouped into two large clusters of the polycistronic type, each borne by one DNA strand and located on each side of a 1.6-kb sequence often termed the switch region. Several hypotheses concerning the role of this switch region have been put forward (region of initiation of transcription for both gene clusters, origin of replication or centromeric sequence). In the present study, we have deleted this region on the three copies of chromosome 1 by sequential targeted replacements. The absence of the switch region did not alter the mitotic stability of the three deleted chromosomes. This region therefore does not appear necessary for chromosomal replication or segregation. However, during the third targeting round, which aimed at knocking out the last switch region, a fourth copy of chromosome 1 that retained this region appeared in all clones analysed. This suggests that the persistence of this switch region is necessary for parasite survival. We then showed that the presence/absence of the switch region did not act upon the expression of a resistance marker gene inserted beforehand into the left gene cluster of the same chromosomal molecule. This result suggests that the presence of this 1.6-kb sequence is not necessary for the expression of all genes on chromosome 1.

Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells.

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.

Parasexuality and mosaic aneuploidy in Leishmania: alternative genetics.

Reproduction as identical or similar organisms in most biological systems depends on the extreme accuracy of the mitotic (and meiotic) mechanisms involved in the transmission of the genetic material to the two daughter cells. Character recombination and genotype diversification are ensured by the alternation between haploidy and diploidy, which corresponds to the most predominant model in sexually reproducing organisms. In Leishmania, the unique association of high levels of automixis and of constitutive 'mosaic aneuploidy' unexpectedly does not lead to loss of heterozygosity but constitutes an alternative for genotype recombination, hence a source of adaptability.

Constitutive mosaic aneuploidy is a unique genetic feature widespread in the Leishmania genus.

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.

The bacterial-like HslVU protease complex subunits are involved in the control of different cell cycle events in trypanosomatids.

The trypanosomatid parasites Leishmania and Trypanosoma are responsible for the most important WHO-designated neglected tropical diseases, for which the need for cost-effective new drugs is urgent. In addition to the classical eukaryotic 20S and 26S proteasomes, these unconventional eukaryotes possess a bacterial-like protease complex, HslVU, made of proteolytic (HslV) and regulatory (HslU) subunits. In trypanosomatids, two paralogous genes are co-expressed: HslU1 and HslU2. Conflicting reports have been published with respect to subcellular localization, functional redundancy and putative roles of the different subunits of this complex in trypanosomatids. Here, we definitively established the mitochondrial localization of HslVU in L. major procyclic promastigotes and of HslV in T. brucei bloodstream trypomastigotes, the latter being the form responsible for the disease in the mammalian host. Moreover, our data demonstrate for the first time the essential nature of HslVU in the bloodstream trypomastigotes of T. brucei, in spite of mitochondrial repression at this stage. Interestingly, our work also allows distinguishing a specific role for the different members of the complex, as HslV and HslU1 appear to be involved in the control of different cell cycle events. Finally, these data validate HslVU as a promising drug target against these parasitic diseases of wide medical and economical importance.

Cell cycle-dependent expression regulation by the proteasome pathway and characterization of the nuclear targeting signal of a Leishmania major Kin-13 kinesin.

The LmjF01.0030 gene of Leishmania major Friedlin, annotated as 'MCAK-like', was confirmed as a kinesin with an internally located motor domain and termed LmjKIN13-1. Both the native form of the protein and a green fluorescent protein (GFP)-fused recombinant version were shown to be exclusively intranuclear, and, more specifically, to localize to the spindle and spindle poles. Cell cycle-dependent regulation of the protein levels was demonstrated using synchronized Leishmania cells: LmjKIN13-1 was highly abundant in the G2+M phase and present at very low levels after mitosis. Altogether, these features suggest that this protein participates in mitosis. The construction of systematic deletion mutants allowed the localization of the primary sequence regions responsible for nuclear targeting on the one hand, and for cell cycle-dependent variations on the other hand. A 42-amino-acid region of the carboxy(C)-terminal domain mediates nuclear import and could be defined as an atypical nuclear localization signal. Protein level regulation during the cell cycle was shown to also depend upon the C-terminal domain, where apparently redundant degradation signals are present. Putative degradation signals appear to be present on both sides and inside the nuclear localization signal. Further experiments strongly suggest a role for the ubiquitin/proteasome pathway in this cell cycle-dependent regulation. These data underline the importance of post-translational regulation of protein abundance in this ancestral eukaryote where transcriptional regulation seems to be rare or near absent.

Comparison of two widely used PCR primer systems for detection of toxoplasma in amniotic fluid, blood, and tissues.

The PCR diagnosis of toxoplasmosis suffers from lack of standardization. Interlaboratory comparative studies of PCR methods have been performed, but intralaboratory comparisons are scarce. Here, we optimized and compared the technical performances of two PCR primer systems widely used for Toxoplasma detection. The differences between the two methods were visible only at low parasite concentrations (< or = 1 Toxoplasma genome per reaction tube). Nevertheless, when clinical samples were tested, both methods significantly differed in their technical sensitivities and specificities. Only one method appeared adequate for samples containing blood or tissue.