The C-terminal segment of Leishmania major HslU: Toward potential inhibitors of LmHslVU activity.

It is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. We explore the possibility to target the parasite mitochondrial HslVU protease, which is essential for growth and has no analogue in the human host. For this, we develop compounds potentially inhibiting the complex assembly by mimicking the C-terminal (C-ter) segment of the ATPase HslU. We previously showed that a dodecapeptide derived from Leishmania major HslU C-ter segment (LmC12-U2, Cpd 1) was able to bind to and activate the digestion of a fluorogenic substrate by LmHslV. Here, we present the study of its structure-activity relationships. By replacing each essential residue with related non-proteinogenic residues, we obtained more potent analogues. In particular, a cyclohexylglycine residue at position 11 (cpd 24) allowed a more than three-fold gain in potency while reducing the size of compound 24 from twelve to six residues (cpd 50) without significant loss of potency, opening the way toward short HslU C-ter peptidomimetics as potential inhibitors of HslV proteolytic function. Finally, conjugates constituted of LmC6-U2 analogues and a mitochondrial penetrating peptide were found to penetrate into the promastigote form of L. infantum and to inhibit the parasite growth without showing toxicity toward human THP-1 cells at the same concentration (i.e. 30 µM).

The HslV Protease from Leishmania major and Its Activation by C-terminal HslU Peptides.

HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as Leishmania. It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from Leishmania major (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.

Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani.

The parasite Leishmania  donovani causes a fatal disease termed visceral leishmaniasis. The process through which the parasite adapts to environmental change remains largely unknown. Here we show that aneuploidy is integral for parasite adaptation and that karyotypic fluctuations allow for selection of beneficial haplotypes, which impact transcriptomic output and correlate with phenotypic variations in proliferation and infectivity. To avoid loss of diversity following karyotype and haplotype selection, L. donovani utilizes two mechanisms: polyclonal selection of beneficial haplotypes to create coexisting subpopulations that preserve the original diversity, and generation of new diversity as aneuploidy-prone chromosomes tolerate higher mutation rates. Our results reveal high aneuploidy turnover and haplotype selection as a unique evolutionary adaptation mechanism that L. donovani uses to preserve genetic diversity under strong selection. This unexplored process may function in other human diseases, including fungal infection and cancer, and stimulate innovative treatment options.

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.

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.

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.

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.

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.

Neuroradiological findings expand the phenotype of OPA1-related mitochondrial dysfunction.

OBJECTIVE: OPA1 mutations are responsible for more than half of autosomal dominant optic atrophy (ADOA), a blinding disease affecting the retinal ganglion neurons. In most patients the clinical presentation is restricted to the optic nerve degeneration, albeit in 20% of them, additional neuro-sensorial symptoms might be associated to the loss of vision, as frequently encountered in mitochondrial diseases. This study describes clinical and neuroradiological features of OPA1 patients. METHODS: Twenty two patients from 17 families with decreased visual acuity related to optic atrophy and carrying an OPA1 mutation were enrolled. Patients underwent neuro-ophthalmological examinations. Brain magnetic resonance imaging (T1, T2 and flair sequences) was performed on a 1.5-Tesla MR Unit. Twenty patients underwent 2-D proton spectroscopic imaging. RESULTS: Brain imaging disclosed abnormalities in 12 patients. Cerebellar atrophy mainly involving the vermis was observed in almost a quarter of the patients; other abnormalities included unspecific white matter hypersignal, hemispheric cortical atrophy, and lactate peak. Neurological examination disclosed one patient with a transient right hand motor deficit and ENT examination revealed hearing impairment in 6 patients. Patients with abnormal MRI were characterized by: (i) an older age (ii) more severe visual impairment with chronic visual acuity deterioration, and (iii) more frequent associated deafness. CONCLUSIONS: Our results demonstrate that brain imaging abnormalities are common in OPA1 patients, even in those with normal neurological examination. Lactate peak, cerebellar and cortical atrophies are consistent with the mitochondrial dysfunction related to OPA1 mutations and might result from widespread neuronal degeneration.

Characterisation of polyglutamylases in trypanosomatids.

Microtubules are subject to post-translational modifications, which are thought to have crucial roles in the function of complex microtubule-based organelles. Among these, polyglutamylation was relatively recently discovered, and was related to centrosome stability, axonemal maintenance and mobility, and neurite outgrowth. In trypanosomatids, parasitic protozoa where microtubules constitute the essential component of the cytoskeleton, the function of polyglutamylated microtubules is unknown. Here, in order to better understand the role of this conserved but highly divergent post-translational modification, we characterised glutamylation and putative polyglutamylases in these parasites. We showed that microtubules are intensely glutamylated in all stages of the cell cycle, including interphase. Moreover, a cell cycle-dependent gradient of glutamylation was observed along the cell anteroposterior axis, which might be related to active growth of the microtubule 'corset' during the cell cycle. We also identified two putative polyglutamylase proteins (among seven analysed here) which appeared to be clearly and directly involved in microtubule polyglutamylation in in vitro activity assays. Paradoxically, in view of the importance of tubulins and of their extensive glutamylation in these organisms, RNA interference-based knockdown of all these proteins had no effect on cell growth, suggesting either functional redundancy or, more likely, subtle roles such as function modulation or interaction with protein partners.